Compositions and methods for delivery of rna

ABSTRACT

The disclosure provides nanoemulsion compositions and methods of making and using thereof to deliver a bioactive agent such as a nucleic acid to a subject. The nanoemulsion composition comprises a hydrophobic core based on inorganic nanoparticles in a lipid nanoparticle that allows imaging as well as delivering nucleic acids. Methods of using these particles for treatment and vaccination are also provided.

This application is a continuation of U.S. application Ser. No.17/839,574, filed Jun. 14, 2022, which is a continuation of U.S.application Ser. No. 17/702,730, filed Mar. 23, 2022, now U.S. Pat. No.11,433,142, issued Sep. 6, 2022, which is a continuation of U.S.application Ser. No. 17/523,457, filed Nov. 10, 2021, now U.S. Pat. No.11,318,213, issued May 3, 2022, which is a continuation of InternationalApplication No. PCT/US2021/019103, filed on Feb. 22, 2021, which claimspriority to U.S. Provisional Application No. 62/993,307, filed on Mar.23, 2020, and U.S. Provisional Application No. 63/054,754, filed on Jul.21, 2020, all of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates generally to RNA delivery. Morespecifically, this invention relates to nanoparticle-mediated deliveryof RNA with a pharmaceutically acceptable nanoparticle that also has theability to be imaged by use of an inorganic reporter inside theparticle.

BACKGROUND

RNA vaccines and therapeutics are a growing area of interest invaccinology and gene therapy. The use of nucleic acid-encoded antigensas the basis for a vaccine platform has numerous advantages:Purification is relatively streamlined and RNA constructs can be builtin days using DNA synthesis technologies followed by RNA transcriptionand capping. This allows for rapid responses to emerging pathogenthreats, pivoting changes in manufacturing to adapt to new circulatingstrains, or for personalizing therapeutic interventions for a variety ofdiseases. While these vaccines and therapies show great promise, in somecases they lack full efficacy in human trials and—like proteinvaccines—may require a method for enhancing their ability to induceadaptive immune responses.

Several approaches have been tested and are in development, but thereremains a need for further and improved nucleic acid vaccines andtherapeutics.

SUMMARY

In brief, the present disclosure provides an inorganic compound-basednanoparticle that binds and delivers RNA to a subject in need oftreatment. This system has numerous advantages: 1) the RNA is deliveredmuch more efficiently than when the RNA is given on its own or whenusing other carrier technologies such as nanostructure lipid carrier; 2)the nanoparticles contain a cationic lipid that stabilizes the RNA andprotects it from degradation; and 3) the nanoparticles have a reporterelement allowing for imaging and tracking the particles in the body.

One aspect of the invention relates to a nanoemulsion compositioncomprising a plurality of nanoemulsion particles. Each nanoemulsionparticle comprises

-   -   a hydrophobic core comprising a mixture of a liquid oil and one        or more inorganic nanoparticles;    -   one or more lipids (such as a cationic lipid); and    -   optionally one or more surfactants.

One aspect of the invention relates to a nanoemulsion compositioncomprising: (i) a plurality of nanoemulsion particles, and (ii) abioactive agent complexed with the nanoemulsion particles. Eachnanoemulsion particle comprises:

-   -   a hydrophobic core comprising a mixture of a liquid oil and one        or more inorganic nanoparticles;    -   one or more lipids (such as a cationic lipid); and    -   optionally one or more surfactants.

Another aspect of the invention relates to a pharmaceutical composition,comprising the nanoemulsion composition comprising the nanoemulsionparticles and the bioactive agent, as described herein, and optionally,a pharmaceutically acceptable carrier or excipient.

Another aspect of the invention relates to a vaccine delivery systemcomprising the nanoemulsion composition comprising the nanoemulsionparticles and the bioactive agent, as described herein, and optionallyone or more vaccine adjuvants, wherein the bioactive agent is an antigenor a nucleic acid molecule encoding an antigen.

Another aspect of the invention relates to a method of delivering abioactive agent to a subject, comprising: administering to the subjectthe nanoemulsion composition comprising the nanoemulsion particles andthe bioactive agent, as described herein.

Another aspect of the invention relates to a method for generating animmune response in a subject, comprising: administering to a subject thenanoemulsion composition comprising the nanoemulsion particles and thebioactive agent, as described herein, and optionally an adjuvant,wherein the bioactive agent is an antigen or a nucleic acid moleculeencoding an antigen.

Another aspect of the invention relates to a method of treating orpreventing an infection or disease in a subject, comprising:administering to the subject a therapeutically effective amount of thenanoemulsion composition comprising the nanoemulsion particles and thebioactive agent, as described herein, and optionally a pharmaceuticallyacceptable carrier.

Another aspect of the invention relates to a method of imaging and/ortracking a bioactive agent delivery in a subject, comprising:

-   -   administering to the subject the nanoemulsion composition        comprising the nanoemulsion particles and the bioactive agent,        as described herein, wherein the inorganic nanoparticles contain        materials detectable via magnetic resonance imaging, and    -   detecting the nanoemulsion composition with magnetic resonance        imaging.

Another aspect of the invention relates to a method of making ananoemulsion composition, comprising:

-   -   (a) mixing one or more inorganic nanoparticles, a liquid oil,        one or more lipids (such as a cationic lipid), and optionally, a        hydrophobic surfactant, thereby forming an oil-phase mixture;    -   (b) mixing the oil-phase mixture with an aqueous solution,        optionally containing a hydrophilic surfactant, to form        nanoemulsion particles; and    -   (c) optionally, mixing the nanoemulsion particles with an        aqueous solution containing a bioactive agent, thereby        complexing the bioactive agent with the nanoemulsion particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the particle size and stability of three exemplarylipid inorganic nanoparticles (LION) formulations at varioustemperatures as a function time. FIG. 1A shows LIONs labeled as 79-004produced in Example 1. FIG. 1B shows LIONs labeled as 79-006-A producedin Example 1. FIG. 1C shows LIONs labeled as 79-006-B produced inExample 1.

FIG. 2 shows the gel electrophoresis of exemplary LION formulations(LIONs labeled as 79-004, 79-006-A, 79-006-B, and 79-011, respectively,as prepared in Example 1) complexed with RNA molecules atnitrogen:phosphate (N:P) ratio of 15, as compared to the naked(unformulated) RNA, demonstrating the ability of the LION formulationsto protect RNA from the action of RNases.

FIG. 3A shows the protein expression in C57BL/6 mice injectedintramuscularly with repRNA-encoding SEAP formulated with an LIONformulation (LION labeled as 79-004, as prepared in Example 1) over aprolonged period of time. FIG. 3B shows the protein expression inC57BL/6 mice injected intramuscularly with repRNA-encoding SEAPformulated with various LION formulations varying SPIO sizes (LIONslabeled as 79-004, 79-006-A, and 79-006-B, respectively, as prepared inExample 1) over days post injection.

FIG. 3C shows the protein expression in C57BL/6 mice injectedintramuscularly with repRNA-encoding SEAP formulated with an LIONformulation, or with a nanostructured lipid carrier (NLC) as control,over days post injection. FIG. 3D shows the protein expression inC57BL/6 mice injected intramuscularly with repRNA-encoding SEAPformulated with an LION formulation at a RNA complex concentration of400 ng/μl and 40 ng/μl, respectively, over days post injection. FIG. 3Eshows the protein expression in C57BL/6 mice injected intramuscularlywith repRNA-encoding SEAP (at 0.5 μg, 2.5 μg, and 12.5 μg, respectively)formulated with an LION formulation as a function of the N:P ratio. Dataare displayed as mean and SE.

FIG. 4A shows that the antigens expressed from the LION/repRNA complex(at the dosage levels of 10, 1, and 0.1 μg of srRNA) induced immuneresponse to the receptor-binding domain of SARS-CoV-2 in C57BL/6 mice.FIG. 4B shows the results of anti-S IgG concentrations in the serum ofthe C57BL/6 mice injected intramuscularly with repSARS-CoV2S formulatedwith various LION formulations varying SPIO size (LION-10, LION-15,LION-25, LION-5 respectively), determined by anti-Spike enzyme linkedimmunosorbent assay (ELISA). Data are displayed as mean and SE; n=5 pergroup. FIG. 4C shows the results of anti-S IgG concentrations in theserum of the C57BL/6 mice injected intramuscularly with repSARS-CoV2Sformulated with various LION formulations by varying the mixingdirection (mixing LION to RNA vs. mixing RNA to LION) and diluent (1:200dilution using sucrose (Suc) vs. using dextrose (Dex)), at Day 14 (firstbar for each group) or Day 21 (second bar for each group) afterintramuscular injection, determined by anti-Spike ELISA. Data aredisplayed as mean and SE; n=5 per group.

FIGS. 5A-5B show the enhancement in T1 (FIG. 5A) and T2 (FIG. 5B)relaxation times as a function of iron concentration in LIONformulations.

FIG. 6A shows the LION-antibody sequence RNA complex induced ZIKV-117antibodies in animals. Animals were bled 7 days after immunization.FIGS. 6B and 6C show the magnitude and kinetics of anti-BG505 SOSIP.664IgG antibodies in adult female pregnant rabbits immunized byintramuscular route with saline (FIG. 6B) or repRNA encoding BG505SOSIP.664 trimer formulated with LION (FIG. 6C). FIGS. 6D and 6E showthe magnitude and kinetics of anti-ZIKV E IgG antibodies in adult femalepregnant rabbits immunized by intramuscular route with saline (FIG. 6D)or repRNA encoding ZIKV prM-E antigens formulated with LION (FIG. 6E).The shaded region around week 1 marks the period when rabbits were bred.The shaded region between weeks 6 and 7 marks the period when kits weredelivered. Arrows mark immunization time points (weeks 0, 4 and 11).FIGS. 6F and 6G show the results for the evaluation of in utero transferof anti-SOSIP IgG from rabbit does to rabbit kits. FIG. 6F showsanti-SOSIP IgG responses in rabbit kits at time of delivery. A minimumof two rabbit kits from each litter per treatment group were euthanizedto evaluate in utero antibody transfer. FIG. 6G shows the XY plotdemonstrating a positive correlation (Pearson r=0.94) between antibodylevels in rabbit does and corresponding rabbit kits. FIGS. 6H and 6Ishow the vaccine-induced responses in the context of pre-existingmaternal antibodies. Serum anti-SOSIP IgG levels were collected inrabbit kits 4 weeks post-boost (3 weeks after kits were weaned). Therabbit kits from rabbit does receiving saline or from rabbit doesreceiving LION+RNA-prM/E are grouped as negative (—) for pre-existingmaternal antibodies against BG505 SOSIP.664. The rabbit kits from rabbitdoes receiving LION+RNA-SOSIP or AddaVax adjuvanted recombinant BG505SOSIP.664 are grouped as positive (+) for pre-existing maternalantibodies against BG505 SOSIP.664. Ordinary one-way ANOVA and Tukey'smultiple comparisons test was performed on log 10 transformed data.(ns=non significant).

FIGS. 7A-7C show the repRNA-CoV2S characterization in vitro. FIG. 7Ashows that codon-optimized full length spike (S) open reading frame,including the S1-, S2-, transmembrane- (TM), and cytoplasmic- (CD)domains, corresponding to positions 21,536 to 25,384 in SARS-CoV-2isolate Wuhan-Hu-1 (GenBank: MN908947.3), fused to a c-terminal v5epitope tag, was cloned into an alphavirus replicon encoding the 4nonstructural protein (nsP1-4) genes of Venezuelan equine encephalitisvirus, strain TC-83. FIGS. 7B and 7C show the analysis results of cells,24 hours later following the transfection of repRNA-COV2S into BHKcells, by anti-v5 immunofluorescence (FIG. 7B) and western blot (FIG.7C), using either convalescent human serum or anti-v5 forimmunodetection. Recombinant SARS-CoV2 spike protein (rCoV2-Spike) andrepRNA-GFP were used as positive and negative controls, respectively.Data in FIGS. 7B and 7C are representative of two independentexperiments.

FIGS. 8A-8E show the exemplary Lipid InOrganic Nanoparticle (LION)formulation of repRNA. FIG. 8A is a graphical representation of anexemplary LION and its formation of vaccine complex after mixing withrepRNA. FIG. 8B is a graph showing the time evolution of LION particlesize, measured by dynamic light scattering (DLS), after storage at 4°C., 25° C. and 42° C. FIG. 8C is a graph showing the confirmation of acomplex formation by a shift in size distribution, after mixing LION andrepRNA. FIG. 8D shows the gel electrophoresis analysis of triplicatepreparations of repRNA extracted from LION, following a concentratedRNase challenge, illustrating substantial protection relative to atriplicate preparation of a dose-matched naked RNA, following a RNAsechallenge. FIG. 8E shows the gel electrophoresis of repRNA extracted byphenol-chloroform treatment. FIG. 8F shows the particle size of thecomplex. Data in FIG. 8B, FIG. 8E, and FIG. 8F are from a singleexperiment, while data in FIG. 8C and FIG. 8D are representative ofthree independent experiments. Data in FIG. 8B, FIG. 8D, and FIG. 8F areshown as mean±s.d. of 3 technical replicates.

FIGS. 9A-9F show that the LION/repRNA-CoV2S complex induced Th1-biasedand neutralizing antibodies in C57BL/6 mice. Six to eight-week oldC57BL/6 mice (n=5/group) received 10, 1, or 0.1 μg LION/repRNA-CoV2S viathe intramuscular route. Fourteen days after prime immunization, serumwas harvested. FIG. 9A shows the results of anti-S IgG concentrations inthe serum of the C57BL/6 mice, determined by enzyme linked immunosorbentassay (ELISA). FIG. 9B shows the 50% inhibitory concentrations (IC50) inthe serum of the C57BL/6 mice, determined by pseudovirus (SARS-CoV-2Wuhan-Hu-1 pseudotype) neutralization assays. FIG. 9C and FIG. 9D showthe anti-S IgG1 and IgG2c concentrations (FIG. 9C) and the IgG2c:IgG1concentration ratio (FIG. 9D) in the serum of the C57BL/6 mice,determined by ELISA. On day 28, mice received a booster immunization,and 12 days later, the spleens and lungs were harvested. FIGS. 9E and 9Fshow the results of the IFN-γ responses in spleen cells (FIG. 9E) and inlung cells (FIG. 9F), measured by enzyme-linked immune absorbent spot(ELISpot), following 18-hour stimulation with 10 peptide poolsencompassing the S protein and consisting of 15-mers overlapping by 11amino acids. Data in FIG. 9A, FIG. 9C, and FIG. 9D are representative ofthree independent experiments, while data in FIG. 9B, FIG. 9E, and FIG.9F were from a single experiment. All data are represented as individualvalues as well as mean±s.d. *p<0.05, as determined by one-way ANOVA withTukey's multiple comparison test.

FIGS. 10A-10C show that the LION/repRNA-CoV2S complex induced Th1-biasedantibodies in aged BALB/C mice. Two-, eight-, or seventeen-month oldBALB/C mice (n-5/group) received 10 or 1 μg LION/repRNA-CoV2S via theintramuscular route. Fourteen days after prime immunization, serum washarvested. FIGS. 10A, 10B, and 10C show the results of the anti-S IgGconcentration (FIG. 10A), IgG1 concentration and IgG2a concentrations(FIG. 10B), and the IgG2a:IgG1 concentration ratios (FIG. 10C) in theserum of the aged BALB/C mice, determined by ELISA. Data in 17-, 8-, and2-month old BALB/Cs were from a single experiment, and data for the2-month old BALB/Cs were replicated in a second experiment. All data arerepresented as individual values as well as mean±s.d. *p<0.05, asdetermined by one-way ANOVA with Tukey's multiple comparison testbetween the 17-month old animals and either the 8- or 2-month oldanimals.

FIGS. 11A-11D show that a single dose of the LION/repRNA-CoV2S complexinduced neutralizing antibody responses in pigtailed macaques. FIG. 11Ashows the dosage regime, in which pigtail macaques were vaccinated with250 μg (n=3) or with 50 μg (n=2) repRNA-CoV2-S complex via theintramuscular route, with the blood being collected on days 10, 14, 28,and 42; the 50 μg group received a boost vaccination on day 28, with theblood being collected 14 days later. FIG. 11B shows the results of theserum anti-S IgG ELISAs performed on the post-immunization samples,against the baseline established by the pre-immunization blood draws.FIG. 11C shows the results of the mean 50% inhibitory concentrations(IC50) of each sample, determined by the pseudovirus (SARS-CoV-2Wuhan-Hu-1 pseudotype) neutralization assays, against the baselineestablished by the pre-immunization blood draws. FIG. 11D shows that 80%plaque-reduction neutralizing antibody titers (PRNT₈₀) againstSARS-CoV2/WA/2020 isolate were measured at days 28 and 42 alongside serafrom 7 convalescent human samples collected from confirmed COVID-19patients. The experiment was performed once. Each line in FIG. 11B andFIG. 11C are representative of each individual animal. Data in FIG. 11Dare reported as individual values as well as mean±s.d. *p<0.05, asdetermined by students t-test comparing 250 μg groups at days 14 and 28.There was no significant difference (ns) between mean PRNT₈₀ titers inall 5 animals at day 42 and titers in sera from 7 convalescent humans,as measured by Mann-Whitney U test.

FIG. 12 shows the anti-spike IgG levels in the rabbits injectedintramuscularly with repRNA-SARS-CoV2S (at 250 μg and 10 μg dose level,respectively) formulated with LION formulation. Rabbits were bled atregular intervals after intramuscular injection, and protein expressionwas determined by assaying IgG concentrations by anti-Spike ELISA. Eachpoint represents data from an individual animal. Data are displayed asmean and SEM, n=4 per group.

FIG. 13A shows the anti-F IgG levels in C57Bl/6 and BALB/c mice injectedintramuscularly with 2.5 μg repRNA-RSV complexed with a LIONformulation. FIG. 13B shows the anti-G (A2) IgG levels in C57Bl/6 andBALB/c mice injected intramuscularly with 2.5 μg repRNA-RSV complexedwith a LION formulation. Blood was collected 28 days after intramuscularinjection, and the serum was prepared and assessed by ELISA. Repliconnumber (645 or 646) is indicated in the parentheses. Each pointrepresents data from an individual animal, with the whiskersrepresenting minimum to maximum, the box representing the interquartilerange and the horizontal bar depicting the median.

FIGS. 14A-14B show the binding of PAMP to the LION formulation thatprovided protection from RNase challenge. FIG. 14A shows the gelelectrophoresis analysis of PAMP-LION complexes at various N:Pcomplexing ratio (0.04, 0.2, 1, 5, and 25, respectively) run on an RNAgel and was assessed for free RNA. FIG. 14B shows the gelelectrophoresis analysis of PAMP-LION complexes, following a challengewith RNase A, as compared to naked PAMP (unformulated PAMP). RNA wasextracted from LION and run on an agarose gel to assess RNA degradation.

FIG. 15 shows the activation of the IFN-β promoter and IFIT2 measured bySEAP activity and luciferase activity in the supernatant, respectively,by the PAMP-LION complex as a function of N:P ratio. Dashed linesrepresent activation levels of PAMP alone.

FIGS. 16A and 16B show the activation of the IFN-β promoter (FIG. 16A)and IFIT2 (FIG. 16B) measured by SEAP activity and luciferase activityin the supernatant, respectively, by the PAMP-LION formulation orRiboxxim-LION formulation, as compared to unformulated RNA. The dashedlines represent OD635 readings of media control wells. FIG. 16C showsthe activation of the IFIT2 by the Riboxxim-LION formulation, ascompared to unformulated Riboxxim, as a function of the Riboxxim doselevel. FIG. 16D shows the dose-dependent induction of innate immunegenes in the nasal cavity of treated mice compared to naïve controls.FIG. 16E shows the activation of innate immune genes in the lungs oftreated mice. FIG. 16F shows that the mice maintained body weight whenbeing administered the PAMP:LION formulation intranasally for 3consecutive days.

FIG. 17 shows in vitro protein expression from exemplary RNA:LIONcomplexes with replicon RNA encoding nLuc, using SPIO (Fe-LION) orTOPO-coated aluminum oxyhydroxide nanoparticles (Al-LION) as the core ofthe LION formulation.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure provides for use of Lipid InOrganic Nanoparticles(LIONs) as carriers of RNA. In particular, a solid inorganic core in alipid matrix with a charged coating in a buffer is disclosed. The use ofthese nanoparticles has numerous advantages: RNA can be complexedindependent of the particles, and the particle can be designed to havemagnetic signals, such as useable for MRI or other imaging techniques.RNA is protected by the particles and they drive expression of numeroustypes of protein including antigens off of the protected RNA when givento cells or a living being.

One aspect of the invention relates to a nanoemulsion compositioncomprising a plurality of nanoemulsion particles. Each nanoemulsionparticle comprises

-   -   a hydrophobic core comprising a mixture of a liquid oil and one        or more inorganic nanoparticles;    -   one or more lipids (e.g., a cationic lipid); and    -   optionally one or more surfactants.

Another aspect of the invention relates to a nanoemulsion compositioncomprising: (i) a plurality of nanoemulsion particles, and (ii) abioactive agent complexed with the nanoemulsion particles. Eachnanoemulsion particle comprises:

-   -   a hydrophobic core comprising a mixture of a liquid oil and one        or more inorganic nanoparticles;    -   one or more lipids (e.g., a cationic lipid); and    -   optionally one or more surfactants.

The Nanoemulsion Particles

The nanoemulsion particle has a hydrophobic core comprising a mixture ofa liquid oil and one or more inorganic solid nanoparticles. Thenanoemulsion particle can also be referred to herein as Lipid InOrganicNanoparticles (LIONs).

The liquid oil is mixed with the one or more inorganic nanoparticles toform a hydrophobic core. The liquid oil is typically metabolizable.Suitable liquid oil can be a vegetable oil, animal oil, or syntheticallyprepared oil.

In some embodiments, the liquid oil is a fish oil. In some embodiments,the liquid oil is a naturally occurring or synthetic terpenoid.

In some embodiments, the liquid oil is squalene, triglyceride (such ascapric/caprylic triglyceride or myristic acid triglyceride), vitamin E,lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, sunfloweroil, soybean oil, olive oil, grapeseed oil, or a combination thereof. Inone embodiment, the liquid oil is squalene, triglyceride (such ascapric/caprylic triglyceride or myristic acid triglyceride), vitamin E,lauroyl polyoxylglyceride, monoacylglycerol, soy lecithin, or acombination thereof. In one embodiment, the liquid oil is squalene,triglyceride (such as capric/caprylic triglyceride or myristic acidtriglyceride), sunflower oil, soybean oil, olive oil, grapeseed oil, ora combination thereof.

In some embodiments, the liquid oil is squalene (either naturallyoccurring or synthetic, optionally in combination with any of the abovelisted liquid oils.

The inorganic nanoparticles may be formed from one or more same ordifferent metals (any metals including transition metal), such as frommetal salts, metal oxides, metal hydroxides, and metal phosphates.Examples include silicon dioxide (SiO₂), iron oxides (Fe₃O₄, Fe₂O₃, FeO,or combinations thereof), aluminum oxide (Al₂O₃), aluminum oxyhydroxide(AlO(OH)), aluminum hydroxyphosphate (Al(OH)_(x)(PO₄)_(y)), calciumphosphate (Ca₃(PO₄)₂), calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), irongluconate, or iron sulfate.

In some embodiments, the inorganic solid nanoparticle is a metal oxide,such as a transition metal oxide. In one embodiment, the inorganic solidnanoparticle is an iron oxide, for instance, magnetite (Fe₃O₄),maghemite (γ-Fe₂O₃), wüstite (FeO), hematite (α-Fe₂O₃), or combinationsthereof.

In some embodiments, the inorganic solid nanoparticle is a metalhydroxide, such as an aluminum hydroxide or aluminum oxyhydroxide.

The inorganic solid nanoparticle may contain a reporter elementdetectable via imaging methods to allow for imaging and tracking theresulting nanoemulsion particles in the body. For instance, theinorganic solid nanoparticle may contain a reporter element detectablevia magnetic resonance imaging (MRI), such as a paramagnetic,superparamagnetic, ferrimagnetic or ferromagnetic compound. Exemplaryinorganic solid nanoparticle materials that are MRI-detectable are ironoxides, iron gluconates, and iron sulfates.

The inorganic solid nanoparticle typically has an average diameter(number weighted average diameter) ranging from about 3 nm to about 50nm. For instance, the inorganic solid nanoparticle can have an averagediameter of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm.

The inorganic solid nanoparticle may be surface modified before mixingwith the liquid oil. For instance, if the surface of the inorganic solidnanoparticle is hydrophilic, the inorganic solid nanoparticle may becoated with hydrophobic molecules (or surfactants) to facilitate themiscibility of the inorganic solid nanoparticle with the liquid oil inthe “oil” phase of the nanoemulsion particle. Phosphate-terminatedlipids (such as phosphatidylated lipids), phosphorous-terminatedsurfactants, carboxylate-terminated surfactants, sulfate-terminatedsurfactants, or amine-terminated surfactants can be used for surfacemodification of the inorganic solid nanoparticle. Typicalphosphate-terminated lipids or phosphorous-terminated surfactants aretrioctylphosphine oxide (TOPO) or distearyl phosphatidic acid (DSPA).Typical sulfate-terminated surfactants include but not limited to sodiumdodecyl sulfate (SDS). Typical carboxylate-terminated surfactantsinclude oleic acid. Typical amine terminated surfactants includeoleylamine.

In one embodiment, the inorganic solid nanoparticle is a metal oxidesuch as an iron oxide, and a surfactant, such as oleic acid, oleylamine,SDS, DSPA, or TOPO, is used to coat the inorganic solid nanoparticle,before it is mixed with the liquid oil to form the hydrophobic core.

In one embodiment, the inorganic solid nanoparticle is a metalhydroxide, such as an aluminum hydroxide or aluminum oxyhydroxide, and aphosphate-terminated lipid or a surfactant, such as oleic acid,oleylamine, SDS, TOPO or DSPA is used to coat the inorganic solidnanoparticle, before it is mixed with the liquid oil to form thehydrophobic core.

The lipids used to form nanoemulsion particles can be cationic lipids,anionic lipids, neutral lipids, or mixtures thereof.

In some embodiments, the lipids used are cationic lipids. For example,positively charged lipids that can have favorable interactions withnegatively charged bioactive agent (such as DNAs or RNAs) may be used inthe nanoemulsion composition. Suitable cationic lipids include1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP);3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DCCholesterol); dimethyldioctadecylammonium (DDA);1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP),dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP);distearoyltrimethylammonium propane (DSTAP);N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC);1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC);1,2-dioleoyl-3-dimethylammonium-propane (DODAP); and1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA);1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C12-200); and combinations thereof. A typical cationic lipid is DOTAP.

Other examples for suitable lipids include, but are not limited to, thephosphatidylcholines (PCs), such as distearoylphosphatidylcholine(DSPC), dioleoyl phosphatidylcholine (DOPC),1-palmitoyl-2-oleoylphosphatidylcholine (POPC),dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylcholine(DMPC), etc.; phosphatidylethanolamines (PEs), such as1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),dioleoylphosphatidylethanolamine (DOPE), etc.; phosphatidylglycerol(PGs); and PEGylated lipids including PEGylated version of any of theabove lipids (e.g., DSPE-PEGs).

The nanoemulsion particle can further contain one or more surfactants,which can be a hydrophobic surfactant or a hydrophilic surfactant. Insome embodiments, the nanoemulsion particle further comprises ahydrophobic surfactant. In some embodiments, the nanoemulsion particlefurther comprises a hydrophilic surfactant. In one embodiment, thenanoemulsion particle further comprises a hydrophobic surfactant and ahydrophilic surfactant.

Suitable hydrophobic surfactants include those having ahydrophilic-lipophilic balance (HLB) value of 10 or less, for instance,5 or less, from 1 to 5, or from 4 to 5. An exemplary hydrophobicsurfactant is a sorbitan ester (such as sorbitan monoester or sorbitantrimester). For instance, the hydrophobic surfactant can be a sorbitanester having a HLB value from 1 to 5, or from 4 to 5.

In some embodiments, the hydrophobic surfactant is a sorbitan monoesteror a sorbitan triester. Exemplary sorbitan monoesters include sorbitanmonostearate and sorbitan monooleate. Exemplary sorbitan triestersinclude sorbitan tristearate and sorbitan trioleate.

Suitable hydrophilic surfactants include those polyethylene oxide-basedsurfactants, for instance, a polyoxyethylene sorbitan ester(polysorbate). In some embodiments, the hydrophilic surfactant is apolysorbate. Exemplary polysorbates are polysorbate 80 (polyoxyethylenesorbitan monooleate, or Tween 80), polysorbate 60 (polyoxyethylenesorbitan monostearate, or Tween 60), polysorbate 40 (polyoxyethylenesorbitan monopalmitate, or Tween 40), and polysorbate 20(polyoxyethylene sorbitan monolaurate, or Tween 20). In one embodiment,the hydrophilic surfactant is polysorbate 80.

The nanoemulsion particle can have an oil-to-surfactant molar ratioranging from about 0.1:1 to about 20:1, from about 0.5:1 to about 12:1,from about 0.5:1 to about 9:1, from about 0.5:1 to about 5:1, from about0.5:1 to about 3:1, or from about 0.5:1 to about 1:1.

The nanoemulsion particle can have a hydrophilic surfactant-to-lipid(e.g., cationic lipid) ratio ranging from about 0.1:1 to about 2:1, fromabout 0.2:1 to about 1.5:1, from about 0.3:1 to about 1:1, from about0.5:1 to about 1:1, or from about 0.6:1 to about 1:1.

The nanoemulsion particle can have a hydrophobic surfactant-to-lipid(e.g., cationic lipid) ratio ranging from about 0.1:1 to about 5:1, fromabout 0.2:1 to about 3:1, from about 0.3:1 to about 2:1, from about0.5:1 to about 2:1, or from about 1:1 to about 2:1.

The nanoemulsion particle can comprise from about 0.2% to about 40% w/vliquid oil, from about 0.001% to about 10% w/v inorganic solidnanoparticle, from about 0.2% to about 10% w/v lipid (e.g., cationiclipid), from about 0.25% to about 5% w/v hydrophobic surfactant (e.g.,sorbitan ester), and from about 0.5% to about 10% w/v hydrophilicsurfactant.

In certain embodiments, the nanoemulsion particle comprises:

-   -   a hydrophobic core comprising a mixture of:        -   one or more inorganic nanoparticles containing at least one            metal oxide nanoparticle optionally coated with a            phosphate-terminated lipid, a phosphorous-terminated            surfactant, a carboxylate-terminated surfactant, a            sulfate-terminated surfactant, or an amine-terminated            surfactant, and a liquid oil containing naturally occurring            or synthetic squalene;        -   a cationic lipid comprising DOTAP;    -   a hydrophobic surfactant comprising a sorbitan ester selected        from the group consisting of sorbitan monostearate, sorbitan        monooleate, and sorbitan trioleate; and    -   a hydrophilic surfactant comprising a polysorbate.

In one embodiment, the nanoemulsion particle comprises:

-   -   a hydrophobic core comprising a mixture of:        -   one or more inorganic nanoparticles containing iron oxide            nanoparticles, and        -   a liquid oil containing naturally occurring or synthetic            squalene;    -   the cationic lipid DOTAP;    -   a hydrophobic surfactant comprising sorbitan monostearate; and    -   a hydrophilic surfactant comprising polysorbate 80.

In this LION composition, the LION particle can comprise from about 0.2%to about 40% w/v squalene, from about 0.001% to about 10% w/v iron oxidenanoparticles, from about 0.2% to about 10% w/v DOTAP, from about 0.25%to about 5% w/v sorbitan monostearate, and from about 0.5% to about 10%w/v polysorbate 80.

In one embodiment, the LION particle comprises from about 2% to about 6%w/v squalene, from about 0.01% to about 1% w/v iron oxide nanoparticles,from about 0.2% to about 1% w/v DOTAP, from about 0.25% to about 1% w/vsorbitan monostearate, and from about 0.5%) to about 5% w/v polysorbate80.

In certain embodiments, the nanoemulsion particle comprises:

-   -   a hydrophobic core comprising a mixture of:        -   one or more inorganic nanoparticles containing at least one            metal hydroxide or oxyhydroxide nanoparticle optionally            coated with a phosphate-terminated lipid, a            phosphorous-terminated surfactant, a carboxylate-terminated            surfactant, a sulfate-terminated surfactant, or an            amine-terminated surfactant, and        -   a liquid oil containing naturally occurring or synthetic            squalene;    -   a cationic lipid comprising DOTAP;    -   a hydrophobic surfactant comprising a sorbitan ester selected        from the group consisting of sorbitan monostearate, sorbitan        monooleate, and sorbitan trioleate; and    -   a hydrophilic surfactant comprising a polysorbate.

In one embodiment, the nanoemulsion particle comprises:

-   -   a hydrophobic core comprising a mixture of:        -   one or more inorganic nanoparticles containing aluminum            hydroxide or aluminum oxyhydroxide nanoparticles optionally            coated with TOPO, and        -   a liquid oil containing naturally occurring or synthetic            squalene;    -   the cationic lipid DOTAP;    -   a hydrophobic surfactant comprising sorbitan monostearate; and    -   a hydrophilic surfactant comprising polysorbate 80.

In this LION composition, the LION particle can comprise from about 0.2%to about 40% w/v squalene, from about 0.001% to about 10% w/v aluminumhydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% toabout 10% w/v DOTAP, from about 0.25% to about 5% w/v sorbitanmonostearate, and from about 0.5% to about 10% w/v polysorbate 80.

In one embodiment, the LION particle comprises from about 2% to about 6%w/v squalene, from about 0.01% to about 1% w/v aluminum hydroxide oraluminum oxyhydroxide nanoparticles, from about 0.2% to about 1% w/vDOTAP, from about 0.25% to about 1% w/v sorbitan monostearate, and fromabout 0.5%) to about 5% w/v polysorbate 80.

Nanoparticles and nanoemulsions have been described in the literatureand the terms are used herein to refer to those particles having a sizeless than 1000 nanometers.

The nanoemulsion particle (LION) typically has an average diameter(z-average hydrodynamic diameter, measured by dynamic light scattering)ranging from about 20 nm to about 200 nm. In some embodiments, thez-average diameter of the LION particle ranges from about 20 nm to about150 nm, from about 20 nm to about 100 nm, from about 20 nm to about 80nm, from about 20 nm to about 60 nm. In some embodiments, the z-averagediameter of the LION particle ranges from about 40 nm to about 200 nm,from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, fromabout 40 nm to about 90 nm, from about 40 nm to about 80 nm, or fromabout 40 nm to about 60 nm. In one embodiment, the z-average diameter ofthe LION particle is from about 40 nm to about 80 nm. In one embodiment,the z-average diameter of the LION particle is from about 40 nm to about60 nm.

The average polydispersity index (PDI) of the nanoemulsion particles(LIONs) can range from about 0.1 to about 0.5. For instance, the averagePDI of the LION particles can range from about 0.2 to about 0.5, fromabout 0.1 to about 0.4, from about 0.2 to about 0.4, from about 0.2 toabout 0.3, or from about 0.1 to about 0.3.

The LION-Bioactive Agent Complex

The nanoemulsion composition can further contain a bioactive agent thatis associated/complexed with the nanoemulsion particles (LIONs). Thebioactive agent may be associated/complexed with the nanoemulsionparticles via non-covalent interactions or via reversible covalentinteractions.

The bioactive agent can be a protein or a bioactive agent encoding aprotein. For instance, the bioactive agent can be a protein antigen or abioactive agent encoding a protein antigen. The antigen can be derivedfrom, or immunologically cross-reactive with, an infectious pathogenand/or an epitope, biomolecule, cell or tissue that is associated withinfection, cancer, or autoimmune disease.

In some embodiments, the bioactive agent is a nucleic acid, such as aRNA or DNA. A variety of RNAs can be associated with the LION particlesfor delivery, including RNAs that modulate innate immune responses, RNAsthat encode proteins or antigens, silencing RNAs, microRNAs, tRNAs,self-replicating RNAs, etc.

In one embodiment, the bioactive agent is mRNA. In one embodiment, thebioactive agent is oncolytic viral RNA. In one embodiment, the bioactiveagent is a replicon RNA.

In certain embodiments, the bioactive agent is an RNA encoding anantigen or an antibody. The antigen may be derived from a bacterialdisease, a viral disease, a protozoan disease, a non-communicabledisease, cancer, or an autoimmune disease. In certain embodiments, theantigen is derived from a RNA virus, such as a hepatitis virus, a coronavirus, a mosquito-borne virus (e.g., Venezuelan equine encephalitis(VEE) virus, or flavivirus such as ZIKV virus), or a HIV virus. Incertain embodiments, the antigen is derived from a corona virus selectedfrom the group consisting a MERS virus and a SARS virus (such asSARS-CoV-2).

In certain embodiments, the bioactive agent is a non-coding RNA.

The bioactive agent can also be an adjuvant. Suitable adjuvants includea TLR agonist, a RIG-I agonist, a saponin, a peptide, a protein, acarbohydrate, a carbohydrate polymer, a conjugated carbohydrate, a wholeviral particle, a virus-like particle, viral fragments, cellularfragments, and combinations thereof.

In certain embodiments, the adjuvant is a TLR agonist or a RIG-Iagonist. Exemplary TLR agonists include a TLR2, TLR3, TLR4, TLR7, TLR8,or TLR9 agonist. A typical TLR agonist is a TLR3 agonist, such asRIBOXXOL, poly(LC), or Hiltonol®.

In certain embodiments, the bioactive agent is a double-stranded RNA.

In certain embodiments, the bioactive agent is an RNA that is an immunestimulator. The immune stimulators can be a TLR3 agonist (e.g., a TLR2,TLR3, TLR4, TLR7, TLR8, or TLR9 agonist) or a RIG-I agonist (e.g., aPAMP). A typical TLR agonist is a TLR3 agonist, such as RIBOXXOL,poly(LC), or Hiltonol®.

As an alternative to, or in addition to the delivery of RNAs asantigens, combinations can be used, e.g., RNA antigens combined withRNAs that stimulate innate immune responses, or RNAs that launchoncolytic viruses, or live-attenuated viruses.

In certain embodiments, the bioactive agent in the nanoemulsioncomposition can comprise a combination of RNA-encoded antigens withanother RNA that can stimulate innate immune responses or can launchoncolytic viruses or live-attenuated viruses. Alternatively, thenanoemulsion composition containing RNA-encoded antigens can be combinedwith a formulation that contains another RNA that can stimulate innateimmune responses or can launch oncolytic viruses or live-attenuatedviruses.

In the nanoemulsion composition, the molar ratio of (i) the nanoemulsionparticles (LIONs) to (ii) the bioactive agent can be characterized bythe nitrogen-to-phosphate molar ratio, which can range from about 0.01:1to about 1000:1, for instance, from about 0.2:1 to about 500:1, fromabout 0.5:1 to about 150:1, from about 1:1 to about 150:1, from about1:1 to about 125:1, from about 1:1 to about 100:1, from about 1:1 toabout 50:1, from about 1:1 to about 50:1, from about 5:1 to about 50:1,from about 5:1 to about 25:1, or from about 10:1 to about 20:1. A molarratio of the nanoemulsion particles (LIONs) to the bioactive agent canbe chosen to increase the delivery efficiency of the bioactive agent,increase the ability of the bioactive agent-carrying nanoemulsioncomposition to elicit an immune response to the antigen, increase theability of the bioactive agent-carrying nanoemulsion composition toelicit the production of antibody titers to the antigen in a subject. Incertain embodiments, the molar ratio of the nanoemulsion particles(LIONs) to the bioactive agent, characterized by thenitrogen-to-phosphate (N:P) molar ratio, ranges from about 1:1 to about150:1, from about 5:1 to about 25:1, or from about 10:1 to about 20:1.In one embodiment, the N:P molar ratio of the nanoemulsion compositionis about 15:1.

By complexing with the bioactive agent, the nanoemulsion composition candeliver the bioactive agent to a cell. The cell can be in a subject inneed. For instance, when the bioactive agent is a protein antigen orencodes a protein antigen, the nanoemulsion composition carrying thebioactive agent can elicit an immune response in the subject against theantigen. The nanoemulsion composition may do so by eliciting antibodytiters to the antigen in the subject, for instance, by inducingneutralizing antibody titers in the subject.

In one embodiment, the nanoemulsion composition containing the LIONs,when administered in an effective amount to the subject, can elicit animmune response to the antigen equal to or greater than the immuneresponse elicited when the bioactive agent is administered to thesubject without the LIONs.

Without being bound by theory, the hydrophobic surfactants in thenanoemulsion composition may contribute to increase the ability of thenanoemulsion composition to deliver a bioactive agent to the cell or toincrease the ability of the nanoemulsion composition carrying abioactive agent to elicit an immune response in the subject against theantigen (when the bioactive agent is a protein antigen or encodes aprotein antigen). For instance, the hydrophobic surfactants in thenanoemulsion composition may contribute to increase the ability of thenanoemulsion composition carrying a bioactive agent

In one embodiment, the hydrophobic surfactant is a sorbitan ester and ispresent in an amount sufficient to increase the ability of thenanoemulsion composition to deliver a bioactive agent to the cell (or tothe subject), as compared to a same nanoemulsion composition, butwithout the sorbitan ester hydrophobic surfactant.

In one embodiment, the hydrophobic surfactant is a sorbitan ester and ispresent in an amount sufficient to increase the ability of the bioactiveagent-carrying nanoemulsion composition to elicit an immune response tothe antigen, as compared to a same nanoemulsion composition, but withoutthe sorbitan ester hydrophobic surfactant.

In one embodiment, the hydrophobic surfactant is a sorbitan ester and,when administered in an effective amount to the subject, thenanoemulsion composition elicits antibody titers to the antigen at ahigher level than the antibody titers elicited when a same nanoemulsioncomposition (but without the sorbitan ester hydrophobic surfactant) isadministered to the subject.

In one embodiment, the hydrophobic surfactant is a sorbitan ester and,when administered in an effective amount to the subject, thenanoemulsion composition induces neutralizing antibody titers in thesubject at a higher level than the neutralizing antibody titers inducedwhen a same nanoemulsion composition (but without the sorbitan esterhydrophobic surfactant) is administered to the subject.

Preparing the Nanoemulsion Composition

Another aspect of the invention relates to a method of making ananoemulsion composition, comprising:

-   -   (a) mixing one or more inorganic nanoparticles, a liquid oil,        one or more lipids (e.g., a cationic lipid), and optionally, a        hydrophobic surfactant, thereby forming an oil phase mixture;        and    -   (b) mixing the oil-phase mixture with an aqueous solution,        optionally containing a hydrophilic surfactant, to form        nanoemulsion particles.

The method can further comprise step (c) mixing the nanoemulsionparticles with an aqueous solution containing a bioactive agent, therebycomplexing the bioactive agent with the nanoemulsion particles.

The bioactive agent may be associated/complexed with the nanoemulsionparticles via non-covalent interactions or via reversible covalentinteractions.

All above descriptions and all embodiments regarding the nanoemulsioncomposition, nanoemulsion particles (including liquid oil, inorganicnanoparticles, lipid such as cationic lipid, hydrophobic surfactant, andhydrophilic surfactant), and bioactive agents discussed above in theaspect of the invention relating to the nanoemulsion compositioncomprising the nanoemulsion particles and in the aspect of the inventionrelating to the nanoemulsion composition comprising the nanoemulsionparticles and a bioactive agent are applicable to this aspect of theinvention.

The resulting nanoemulsion composition can be prepared in a diluted orconcentrated form.

In certain embodiments, the nanoemulsion composition may be diluted (byany suitable buffer solutions) to about 1 to about 200 fold, forinstance, about 1 to about 100 fold, about 2 to about 50 fold, about 2to about 30 fold, about 2 to about 20 fold, about 2 to about 10 fold,about 2 to about 5 fold. In one embodiments, the nanoemulsioncomposition is diluted in 2 fold.

In certain embodiments, the nanoemulsion composition may be concentratedabout 1 to about 100 fold, for instance, about 2 to about 50 fold, about2 to about 30 fold, about 2 to about 20 fold, about 2 to about 10 fold,about 2 to about 5 fold.

The nanoemulsion composition can have a loading capacity for thebioactive agent (e.g., a nucleic acid such as RNA or DNA) of at leastabout 100 μg/ml.

The dosage level of the bioactive agent (e.g., a nucleic acid such asRNA or DNA) in the nanoemulsion composition can range from about 0.001μg/ml to about 1000 μg/ml, for instance, from about 0.002 μg/ml to about500 μg/ml, from about 1 μg/ml to about 500 μg/ml, from about 2 μg/ml toabout 400 μg/ml, from about 40 μg/ml to about 400 μg/ml, or from about10 μg/ml to about 250 μg/ml.

Use of the Nanoemulsion Composition

Various aspects the invention also relate to the use of the nanoemulsioncomposition comprising the nanoemulsion particles and the bioactiveagent, including, for instance, in a pharmaceutical composition, as avaccine delivery system, in delivering a bioactive agent to a cell or asubject, generating an immune response in a subject, and treating asubject in need.

In one aspect, the invention provides a pharmaceutical compositioncomprising the nanoemulsion composition comprising the nanoemulsionparticles and the bioactive agent, as described herein. Optionally, thepharmaceutical composition can comprise a pharmaceutically acceptablecarrier or excipient. As used herein the term “pharmaceuticallyacceptable carrier or excipient” includes solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, etc., compatible with pharmaceutical administration.

In another aspect, the invention provides a vaccine delivery systemcomprising the nanoemulsion composition comprising the nanoemulsionparticles and the bioactive agent, as described herein, and optionallyone or more vaccine adjuvant, wherein the bioactive agent is an antigenor a nucleic acid molecule encoding an antigen.

All above descriptions and all embodiments regarding the nanoemulsioncomposition, nanoemulsion particles (including liquid oil, inorganicnanoparticles, lipid such as cationic lipid, hydrophobic surfactant, andhydrophilic surfactant), bioactive agents, and preparation of thenanoemulsion composition discussed above in the aspect of the inventionrelating to the nanoemulsion composition comprising the nanoemulsionparticles, in the aspect of the invention relating to the nanoemulsioncomposition comprising the nanoemulsion particles and a bioactive agent,and in the aspect of the invention relating to the method of making ananoemulsion composition are applicable to these two aspects of theinvention relating to the pharmaceutical composition and the vaccinedelivery system.

The pharmaceutical composition and the vaccine delivery system can beformulated for various administrative routes, including oraladministration, or parenteral administration, such as intravenous,intramuscular, intradermal, subcutaneous, intraocular, intranasal,pulmonary (e.g., by inhalation) intraperitoneal, or intrarectaladministration.

In one embodiment, the delivery route is pulmonary delivery (e.g., tolung), which can be achieved by different approaches, including the useof nebulized, aerosolized, micellular, or dry powder-based formulations.In one embodiment, the pharmaceutical composition or the vaccinedelivery system are formulated to be administrated in liquid nebulizers,aerosol-based inhalers, and/or dry powder dispersion devices.

One aspect of the invention relates to a method of delivering abioactive agent to a cell, comprising: contacting the cell with thenanoemulsion composition comprising the nanoemulsion particles and thebioactive agent, as described herein.

One aspect of the invention relates to a method of delivering abioactive agent to a subject, comprising: administering to the subjectthe nanoemulsion composition comprising the nanoemulsion particles andthe bioactive agent, as described herein.

All above descriptions and all embodiments regarding the nanoemulsioncomposition, nanoemulsion particles (including liquid oil, inorganicnanoparticles, lipid such as cationic lipid, hydrophobic surfactant, andhydrophilic surfactant), bioactive agents, and preparation of thenanoemulsion composition discussed above in the aspect of the inventionrelating to the nanoemulsion composition comprising the nanoemulsionparticles, in the aspect of the invention relating to the nanoemulsioncomposition comprising the nanoemulsion particles and a bioactive agent,and in the aspect of the invention relating to the method of making ananoemulsion composition are applicable to these two aspects of theinvention relating to the method of delivering a bioactive agent.

The ability and efficiency of the delivery of the bioactive agent by thenanoemulsion particles to a cell or a subject can be controlled byadjusting the components of the nanoemulsion particles, selecting themolar ratio of the nanoemulsion particles (LIONs) to the bioactiveagent, and/or selecting the dosage of the bioactive agent, as describedherein.

One aspect of the invention relates to a method for generating an immuneresponse in a subject, comprising: administering to a subject thenanoemulsion composition comprising the nanoemulsion particles and thebioactive agent, as described herein, and optionally an adjuvant,wherein the bioactive agent is an antigen or a nucleic acid moleculeencoding an antigen.

One aspect of the invention relates to a method of generating an immuneresponse in a subject, comprising:

-   -   (a) administering to the subject a therapeutically effective        amount of an oncolytic virus encoding a protein antigen, and;    -   (b) administering to the subject a therapeutically effective        amount of the nanoemulsion composition comprising the        nanoemulsion particles and the bioactive agent, as described        herein, and optionally an adjuvant, wherein the bioactive agent        is the protein antigen or a nucleic acid molecule encoding the        protein antigen.

All above descriptions and all embodiments regarding the nanoemulsioncomposition, nanoemulsion particles (including liquid oil, inorganicnanoparticles, lipid such as cationic lipid, hydrophobic surfactant, andhydrophilic surfactant), bioactive agents, preparation of thenanoemulsion composition, pharmaceutical composition, and vaccinedelivery system discussed above in the aspects of the invention relatingto the nanoemulsion composition comprising the nanoemulsion particles,relating to the nanoemulsion composition comprising the nanoemulsionparticles and a bioactive agent, relating to the method of making ananoemulsion composition, relating to the pharmaceutical composition,and relating to the vaccine delivery system are applicable to these twoaspects of the invention relating to the method of generating an immuneresponse.

The administrative routes in these methods are the same as thosedescribed above for administrating the pharmaceutical composition andthe vaccine delivery system.

The administration of (a) step and the administration of (b) step canoccur simultaneously. Alternatively, the administration of (a) step andthe administration of (b) step can occur at least 1 week, at least 2weeks, at least 3 weeks, at least 1 month, at least 6 weeks, at leasttwo months, at least three months, at least 6 months, or at least 1 yearapart.

One aspect of the invention also relates to a method of treating orpreventing an infection or disease in a subject, comprising:administering to the subject a therapeutically effective amount of thenanoemulsion composition comprising the nanoemulsion particles and thebioactive agent, as described herein, and optionally a pharmaceuticallyacceptable carrier.

All above descriptions and all embodiments regarding the nanoemulsioncomposition, nanoemulsion particles (including liquid oil, inorganicnanoparticles, lipid such as cationic lipid, hydrophobic surfactant, andhydrophilic surfactant), bioactive agents, preparation of thenanoemulsion composition, pharmaceutical composition, and vaccinedelivery system discussed above in the aspects of the invention relatingto the nanoemulsion composition comprising the nanoemulsion particles,relating to the nanoemulsion composition comprising the nanoemulsionparticles and a bioactive agent, relating to the method of making ananoemulsion composition, relating to the pharmaceutical composition,and relating to the vaccine delivery system are applicable to thisaspect of the invention relating to the method of treating or preventingan infection or disease.

The administrative routes in these methods are the same as thosedescribed above for administrating the pharmaceutical composition andthe vaccine delivery system.

The infection or disease to be treated may be a bacterialinfection/disease, a viral infection/disease, a protozoan disease, anon-communicable disease, cancer, or an autoimmune disease. In someembodiments, the infection/disease is a viral infection/disease causedby an RNA virus. The RNA virus can be a hepatitis virus, a corona virus,a mosquito-borne virus (e.g., Venezuelan equine encephalitis (VEE)virus, or flavivirus such as ZIKV virus), or HIV. To prevent or treatthese diseases, the bioactive agent in the nanoemulsion composition canbe an antigen or a nucleic acid molecule encoding an antigen derivedfrom a corona virus genome.

In certain embodiments, the RNA virus is a corona virus selected fromthe group consisting a MERS virus and a SARS virus. In one embodiment,the SARS virus is SARS-CoV-2.

In some embodiments, the method relates to treating or preventing acorona virus (such as SARS-CoV-2, “COVID-19”) in a subject, and themethod comprises:

-   -   administering to the subject a therapeutically effective amount        of the nanoemulsion composition comprising the nanoemulsion        particles and the bioactive agent, and optionally an adjuvant,        wherein the bioactive agent is: an RNA that is an innate        agonist, or an antigen or a nucleic acid molecule encoding an        antigen derived from a corona virus genome (e.g., the SARS-CoV-2        genome).

In certain embodiments, the bioactive agent is an RNA that is an innateagonist. In one embodiment, the RNA is a RIG-I agonist, such as a PAMP.In one embodiment, the RNA is a TLR3 agonist, such as RIBOXXOL,poly(LC), or Hiltonol®.

In certain embodiments, the bioactive agent is an RNA encoding anantigen derived from the corona virus genome (e.g., the SARS-CoV-2genome). In one embodiment, the RNA is self-replicating. In oneembodiment, the RNA encodes all or a portion of the spike “S” protein.

As discussed above, the inorganic solid nanoparticles, when containing areporter element detectable via imaging methods, the resultingnanoemulsion particles can be imaged and tracked after the nanoemulsionparticles are administered in the body. For instance, the inorganicsolid nanoparticle may contain a reporter element detectable viamagnetic resonance imaging (MRI), such as a paramagnetic,superparamagnetic, ferrimagnetic or ferromagnetic compound.

Accordingly, one aspect of the invention also relates to a method ofimaging and/or tracking a bioactive agent delivery in a subject,comprising:

-   -   administering to the subject the nanoemulsion composition        comprising the nanoemulsion particles and the bioactive agent,        as described herein, wherein the inorganic nanoparticles contain        materials detectable via magnetic resonance imaging, and    -   detecting the nanoemulsion composition with magnetic resonance        imaging.

In one embodiment, the inorganic solid nanoparticle materials that areMRI-detectable are iron oxides, iron gluconates, and iron sulfates.

All above descriptions and all embodiments regarding the nanoemulsioncomposition, nanoemulsion particles (including liquid oil, inorganicnanoparticles, lipid such as cationic lipid, hydrophobic surfactant, andhydrophilic surfactant), bioactive agents, preparation of thenanoemulsion composition, pharmaceutical composition, and vaccinedelivery system discussed above in the aspects of the invention relatingto the nanoemulsion composition comprising the nanoemulsion particles,relating to the nanoemulsion composition comprising the nanoemulsionparticles and a bioactive agent, relating to the method of making ananoemulsion composition, relating to the pharmaceutical composition,and relating to the vaccine delivery system are applicable to thisaspect of the invention relating to the method of imaging and/ortracking a bioactive agent delivery.

The imaging applications of the nanoemulsion compositions are veryuseful as they allow for real-time tracking the delivery of thebioactive agent by the nanoemulsion compositions (LION particles) in thesubject. LIONs therefore not only can deliver the therapy, but also canself-report/tracking the disease and treatment through imaging.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In case ofconflict, the present specification, including explanations of terms,will control. In addition, all the materials, methods, and examples areillustrative and not intended to be limiting.

EXAMPLES

The following examples are for illustrative purposes only and are notintended to limit, in any way, the scope of the present invention.

Example 1. General Production Techniques and Materials Employed in theExamples Materials

The following materials were used in the manufacturing oflipid-inorganic nanoparticles (LIONs). Iron oxide nanoparticles at 25mgFe/ml in chloroform and of various average diameters (5, 10, 15, 20,25 and 30 nm) were purchased from Ocean Nanotech (San Diego, CA).Squalene and Span® 60 (sorbitan monostearate) were purchased fromMillipore Sigma. Tween® 80 (polyethylene glycol sorbitan monooleate) andsodium citrate dihydrate were purchased from Fisher Chemical. Thechloride salt of the cationic lipid1,2-dioleoyl-3-trimethylammonium-propane (DOTAP chloride) was purchasedfrom Corden Pharma. Ultrapure water (18.2 MOhm-cm resistivity) wasobtained from a Milli-Q water purification system (Millipore Sigma).

Production of Lipid Inorganic Nanoparticles (LIONs) Labeled as 79-004.

These LIONs comprise 37.5 mg/ml squalene, 37 mg/ml Span® 60, 37 mg/mlTween® 80, 30 mg/ml DOTAP chloride, 0.1 mg/ml 10 nm iron oxidenanoparticles and 10 mM sodium citrate dihydrate. The LIONs weremanufactured using the following procedures.

In a 200 ml beaker, 0.4 ml of iron oxide nanoparticles at 25 mgFe/ml inchloroform, with a number-weighted average diameter of 10 nm, wereadded. Chloroform was allowed to evaporate in a fume hood leaving behinda dry coating of iron oxide nanoparticles. To the iron oxidenanoparticles, 3.7 grams of Span® 60, 3.75 grams of squalene, and 3grams of DOTAP chloride were added to prepare the “oil” phase. The oilphase was sonicated 30 minutes in a water bath pre-heated to 60° C.Separately, in a 1 liter glass bottle, the “aqueous” phase was preparedby adding 39 grams of Tween® 80 to 1,000 ml 10 mM sodium citratedihydrate solution prepared with Milli-Q water. The aqueous phase wasstirred for 30 minutes to allow complete dissolution of Tween® 80. Aftercomplete dissolution of Tween® 80, 96 ml of the aqueous phase wastransferred to a 200 ml beaker and incubated in a water bath pre-heatedto 60° C. To the heated oil phase, 96 ml of the pre-heated aqueous phasewas added. The mixture was immediately emulsified using a VWR® 200homogenizer (VWR International) until a homogenous colloid with amilk-like appearance was produced. The colloid was subsequentlyprocessed by passaging the fluid through a Y-type interaction chamber ofa LM10 microfluidizer at 20,000 psi. The fluid was passaged until thez-average hydrodynamic diameter, measured by dynamic light scattering(Malvern Zetasizer Nano S), was 54 nm with a 0.2 polydispersity index.The microfluidized LION sample was terminally filtered with a 200 nmpore-size polyethersulfone (PES) syringe filter.

Production of Lipid Inorganic Nanoparticles (LIONs) Labeled as 79-006-A.

These LIONs comprise 37.5 mg/ml squalene, 37 mg/ml Span® 60, 37 mg/mlTween® 80, 30 mg/ml DOTAP chloride, 0.2 mg/ml 15 nm iron oxidenanoparticles, and 10 mM sodium citrate dihydrate. The LIONs weremanufactured using the following procedures.

In a 200 ml beaker, 0.8 ml of iron oxide nanoparticles at 25 mgFe/ml inchloroform, with a number-weighted average diameter of 15 nm, was added.Chloroform was allowed to evaporate in a fume hood leaving behind a drycoating of iron oxide nanoparticles. To the iron oxide nanoparticles,3.7 grams of Span® 60, 3.75 grams of squalene, and 3 grams of DOTAPchloride were added to prepare the “oil” phase. The oil phase wassonicated 30 minutes in a water bath pre-heated to 60° C. Separately, ina 1 liter glass bottle, the “aqueous” phase was prepared by adding 39grams of Tween® 80 to 1,000 ml of 10 mM sodium citrate dihydratesolution prepared with Milli-Q water. The aqueous phase was stirred for30 minutes to allow complete dissolution of Tween® 80. After completedissolution of Tween® 80, 96 ml of the aqueous phase was transferred toa 200 ml beaker and incubated in a water bath pre-heated to 60° C. Tothe heated oil phase, 96 ml of the pre-heated aqueous phase was added.The mixture was immediately emulsified using a VWR® 200 homogenizer (VWRInternational) until a homogenous colloid with a milk-like appearancewas produced. The colloid was subsequently processed by passaging thefluid through a Y-type interaction chamber of a LM10 microfluidizer at20,000 psi. The fluid was passaged until the z-average hydrodynamicdiameter, measured by dynamic light scattering (Malvern Zetasizer NanoS), was 52 nm with a 0.2 polydispersity index. The microfluidized LIONsample was terminally filtered with a 200 nm pore-size polyethersulfone(PES) syringe filter.

Production of Lipid Inorganic Nanoparticles (LIONs) Labeled as 79-006-B.

These LIONs comprise 37.5 mg/ml squalene, 37 mg/ml Span® 60, 37 mg/mlTween® 80, 30 mg/ml DOTAP chloride, 0.2 mg/ml 5 nm iron oxidenanoparticles, and 10 mM sodium citrate dihydrate. The LIONs weremanufactured using the following procedures.

In a 200 ml beaker, 0.8 ml of iron oxide nanoparticles at 25 mgFe/ml inchloroform, with a number-weighted average diameter of 5 nm, was added.Chloroform was allowed to evaporate in a fume hood leaving behind a drycoating of iron oxide nanoparticles. To the iron oxide nanoparticles,3.7 grams of Span® 60, 3.75 grams of squalene, and 3 grams of DOTAPchloride were added to prepare the “oil” phase. The oil phase wassonicated 30 minutes in a water bath pre-heated to 60° C. Separately, ina 1 liter glass bottle, the “aqueous” phase was prepared by adding 39grams of Tween® 80 to 1,000 ml of 10 mM sodium citrate dihydratesolution prepared with Milli-Q water. The aqueous phase was stirred for30 minutes to allow complete dissolution of Tween® 80. After completedissolution of Tween® 80, 96 ml of the aqueous phase was transferred toa 200 ml beaker and incubated in a water bath pre-heated to 60° C. Tothe heated oil phase, 96 ml of the pre-heated aqueous phase was added.The mixture was immediately emulsified using a VWR® 200 homogenizer (VWRInternational) until a homogenous colloid with a milk-like appearancewas produced. The colloid was subsequently processed by passaging thefluid through a Y-type interaction chamber of a LM10 microfluidizer at20,000 psi. The fluid was passaged until the z-average hydrodynamicdiameter, measured by dynamic light scattering (Malvern Zetasizer NanoS), was 59 nm with a 0.2 polydispersity index. The microfluidized LIONsample was terminally filtered with a 200 nm pore-size polyethersulfone(PES) syringe filter.

Production of Lipid Inorganic Nanoparticles (LIONs) Labeled as 79-011.

These LIONs comprise 9.4 mg/ml squalene, 9.3 mg/ml Span® 60, 9.3 mg/mlTween® 80, 7.5 mg/ml DOTAP chloride, 0.05 mg/ml 25 nm iron oxidenanoparticles, and 10 mM sodium citrate dihydrate. The LIONs weremanufactured using the following procedures.

In a 200 ml beaker, 0.2 ml of iron oxide nanoparticles at 25 mgFe/ml inchloroform, with a number-weighted average diameter of 25 nm, was added.Chloroform was allowed to evaporate in a fume hood leaving behind a drycoating of iron oxide nanoparticles. To the iron oxide nanoparticles,0.93 grams of Span® 60, 0.94 grams of squalene, and 0.75 grams of DOTAPchloride were added to prepare the “oil” phase. The oil phase wassonicated 30 minutes in a water bath pre-heated to 60° C. Separately, ina 1 liter glass bottle, the “aqueous” phase was prepared by adding 10grams of Tween® 80 to 1,000 ml of 10 mM sodium citrate dihydratesolution prepared with Milli-Q water. The aqueous phase was stirred for30 minutes to allow complete dissolution of Tween® 80. After completedissolution of Tween® 80, 99 ml of the aqueous phase was transferred toa 200 ml beaker and incubated in a water bath pre-heated to 60° C. Tothe heated oil phase, 96 ml of the pre-heated aqueous phase was added.The mixture was immediately emulsified using a VWR® 200 homogenizer (VWRInternational) until a homogenous colloid with a milk-like appearancewas produced. The colloid was subsequently processed by passaging thefluid through a Y-type interaction chamber of a LM10 microfluidizer at20,000 psi. The fluid was passaged until the z-average hydrodynamicdiameter, measured by dynamic light scattering (Malvern Zetasizer NanoS), was 60 nm with a 0.2 polydispersity index. The microfluidized LIONsample was terminally filtered with a 200 nm pore-size polyethersulfone(PES) syringe filter.

Production of Lipid Inorganic Nanoparticles (LIONs) Labeled as 79-014-A.

These LIONs comprise 9.4 mg/ml squalene, 0.63 mg/ml Dynasan® 114(trimyristin), 9.3 mg/ml Span® 60, 9.3 mg/ml Tween® 80, 7.5 mg/ml DOTAPchloride, 0.05 mg/ml 15 nm iron oxide nanoparticles, and 10 mM sodiumcitrate dihydrate. The LIONs were manufactured using the followingprocedures.

In a 200 ml beaker, 0.2 ml of iron oxide nanoparticles at 25 mgFe/ml inchloroform, with a number-weighted average diameter of 15 nm, was added.Chloroform was allowed to evaporate in a fume hood leaving behind a drycoating of iron oxide nanoparticles. To the iron oxide nanoparticles,0.93 grams of Span® 60, 0.94 grams of squalene, 0.063 grams Dynasan®114, and 0.75 grams of DOTAP chloride were added to prepare the “oil”phase. The oil phase was sonicated 30 minutes in a water bath pre-heatedto 60° C. Separately, in a 1 liter glass bottle, the “aqueous” phase wasprepared by adding 10 grams of Tween® 80 to 1,000 ml of 10 mM sodiumcitrate dihydrate solution prepared with Milli-Q water. The aqueousphase was stirred for 30 minutes to allow complete dissolution of Tween®80. After complete dissolution of Tween® 80, 99 ml of the aqueous phasewas transferred to a 200 ml beaker and incubated in a water bathpre-heated to 60° C. To the heated oil phase, 96 ml of the pre-heatedaqueous phase was added. The mixture was immediately emulsified using aVWR® 200 homogenizer (VWR International) until a homogenous colloid witha milk-like appearance was produced. The colloid was subsequentlyprocessed by passaging the fluid through a Y-type interaction chamber ofa LM10 microfluidizer at 20,000 psi. The fluid was passaged until thez-average hydrodynamic diameter, measured by dynamic light scattering(Malvern Zetasizer Nano S), was 60 nm with a 0.2 polydispersity index.The microfluidized LION sample was terminally filtered with a 200 nmpore-size polyethersulfone (PES) syringe filter.

Example 2. Stability of the LION Formulations LION Formulations areThermostable.

Various formulations (LIONs labeled as 79-004, 79-006-A, and 79-006-B,respectively, as prepared in Example 1) were placed into a stabilitychamber at the indicated temperatures. The stability was determined byparticle size measurement using dynamic light scattering. The resultsshow that the LION formulation formed a stable colloid when stored at 4,25 and 42° C. As demonstrated in FIGS. 1A-1C, the particles in the LIONformulations show exceptional stability over a range of temperature andover time.

LION Formulations Protect RNA from RNases.

This example shows that LION formulations protect RNAs from ribonuclease(RNase)-catalyzed degradation. The protection from RNase challenge wascharacterized by gel electrophoresis. RNA molecules were complexed withthe LION formulations by mixing at a predetermined nitrogen:phosphate(N:P) ratio. Various LION formulations were bound to RNA and thecomplexes were exposed to RNase.

One hundred μl of naked (unformulated) RNA or RNA complexed with LIONformulations (LIONs labeled as 79-004, 79-006-A, 79-006-B, and 79-011,respectively, as prepared in Example 1) at N:P of 15 was incubated atroom temperature for 30 minutes with RNase A solution (ThermoScientific, EN053L) diluted to 10 mg/L. After 30 minutes, proteinase Ksolution (Thermo Scientific, E00491) diluted to 1 mg/ml was added andall samples were heated to 55° C. for 10 minutes. To extract RNA, 0.12ml phenol:chloroform solution (Invitrogen, 15593-031) was added to allsamples; samples were vortexed for 15 seconds and centrifuged at 13,300rpm for 15 minutes. 20 μl of supernatant was extracted and transferredto a PCR tube, and 20 μl of glyoxal load dye (Invitrogen, AM8551) wasadded to each tube. All samples were heated at 50° C. for 20 minutes.Samples containing 250 ng of RNA were loaded in the wells of a 1%agarose gel immersed in a Northern Max Gly Gel Prep running buffer(Ambion, AM8678) in a gel electrophoresis box. Gel was run at 120 V for45 minutes and imaged in a gel documentation system. The results areshown in FIG. 2 .

FIG. 2 shows the gel electrophoresis of exemplary LION formulations(LIONs labeled as 79-004, 79-006-A, 79-006-B, and 79-011, respectively,as prepared in Example 1) complexed with RNA molecules atnitrogen:phosphate (N:P) ratio of 15, as compared to the naked(unformulated) RNA. FIG. 2 demonstrates the stability of RNA protectedby the LION formulation to the action of RNase. As shown in FIG. 2 , thenaked RNA (without complexing with a LION formulation) treated withRNase was completely destroyed, while all the LION formulationsprotected the RNA being complexed with.

Example 3. Using LION Formulations for Protein Expression—SecretedEmbryonic Alkaline Phosphatase “SEAP” Expression

This example demonstrates that the LION formulations drive high levelsof Secreted Embryonic Alkaline Phosphatase (SEAP) expression. MessengerRNA molecules encoding a protein of interest were complexed with a LIONformulation, which was delivered in the cytoplasm of cells of anorganism. The messenger RNA underwent intracellular translation andproduced the protein of interest. The subgenome of the attenuatedreplicating alphavirus Venezuelan equine encephalitis (VEE) virus,TC-83, was modified by substituting secreted embryonic alkalinephosphatase (SEAP) for VEE structural proteins.

One μg of the modified replicon RNA encoding SEAP (RNA-SEAP) wascomplexed with LION formulation labeled as 79-004 (10 nm, Example 1) atN:P of 15 and administered intramuscularly (50 μl) in C57BL/6 mice(n=3). Mice were bled at regular intervals, and protein expression wasdetermined by assaying mouse sera for SEAP on days −1 (pre-injection),3, 5, 7, 9, 11, 13, 15 and 20. The results of SEAP expression in mouseover days post injection are shown in FIG. 3A.

FIG. 3A shows that LION formulations complexed with messenger RNAs canexpress robust levels of secreted protein over a prolonged stretch oftime (20 days after injection).

Impact of the Size of the Core Inorganic Particles

RepRNA encoding-SEAP (1 μg) was complexed with LION formulation labeledas 79-004, 79-006-A, or 79-006-B (with varying SPIO sizes, seeExample 1) at a N:P ratio of 15 and administered intramuscularly (50 μl)in C57BL/6 mice (n=3/formulation). Mice were bled at regular intervals,and protein expression was determined by assaying mouse sera for SEAP ondays 4, 6, 8, 11, 13, 15 and 20. The results of SEAP expression in mouseover days post injection are shown in FIG. 3B. Log-transformed data wasanalyzed by two-way ANOVA and Tukey's multiple comparisons test.

FIG. 3B shows that LION formulations with the core inorganic SPIOnanoparticles having varying average diameters all worked to inducesimilar robust levels of protein expression in vivo over a prolongedtime period, with no statistically significant difference in SEAPexpression between there formulations. Thus, the diameter of SPIOparticles did not appear to impact the ability of the LION formulationto deliver repRNA molecules and the level of protein expression from therepRNA molecules.

LION Delivery vs. NLC Delivery

The modified repRNA-encoding SEAP (RNA-SEAP) (1 μg) was complexed withLION formulation labeled as 79-004 (10 nm, Example 1) at N:P of 15, orwith a nanostructured lipid carrier (NLC) as control. The resultingRNA-LION formulation was administered intramuscularly (50 μl) in C57BL/6mice (n=3). Mice were bled at regular intervals, and protein expressionwas determined by assaying mouse sera for SEAP. The results of SEAPexpression in mouse over days post injection are shown in FIG. 3C.

The control is nanostructured lipid carrier (NLC) which is a blend ofsolid lipid (glyceryl trimyristate-dynasan) and liquid oil (squalene)that forms a semi-crystalline core upon emulsification. See moredetailed description about the NLC in Erasmus et al., “A NanostructuredLipid Carrier for Delivery of a Replicating Viral RNA Provides Single,Low-Dose Protection against Zika,” Mol. Ther. 26(10):2507-22 (2018),which is incorporated herein by reference in its entirety.

As shown in FIG. 3C, the repRNA-encoding SEAP complexed with LIONformulations resulted in higher overall levels of SEAP expression ateach time point, as compared to the repRNA-encoding SEAP complexed withNLC. This indicates that compared to NLC, LION formulation served as abetter delivery vehicle for repRNA molecules. Also, as shown in FIG. 3C,SEAP expression peaked around day 8, and by day 21, it returned tobaseline for the control NLC group and to 0.5 log of baseline for theLION group.

Impact of the RNA Complexing Concentrations

RepRNA encoding-SEAP was complexed with LION formulation (15 nm, similarto 79-006A in Example 1) at a N:P ratio of 15, with varying thecomplexing concentration. The resulting RNA-LION formulations wereadministered intramuscularly in C57BL/6 mice (n=5/formulation). Micewere bled at regular intervals after intramuscular injection, andprotein expression was determined by assaying mouse sera. The results ofSEAP expression in mouse over days post injection are shown in FIG. 3D.

The RNA complexing concentration affected the size of the LION/repRNAcomplex. The LION/repRNA complex having a 10-fold higher repRNAconcentration (400 ng/μl vs. 40 ng/μl) resulted in about 41% largerLION/repRNA-SEAP complex and 24% wider size distribution.

As shown in FIG. 3D, there was no significant difference in the meanSEAP concentration at each time point (using Mann-Whitney test) for theLION/repRNA complexes having concentrations of 400 ng/μl vs. 40 ng/μl,suggesting that the complexing concentration did not have a substantialeffect on the repRNA delivery and protein expression. For either repRNAconcentration (400 ng/μl vs. 40 ng/μl), the SEAP expressionconcentration in the serum of mice immunized with the LION/repRNA-SEAPformulation peaked on Day 7 post intramuscular injection, and returnedto background levels by Day 21.

Impact of the N:P and RNA Dose

RepRNA encoding-SEAP (at 0.5 μg, 2.5 μg, and 12.5 μg, respectively) wascomplexed with LION formulation (15 nm, similar to 79-006A in Example 1)with varying the N:P ratio. The resulting RNA-LION formulations wereadministered intramuscularly in C57BL/6 mice (n=4/formulation). Micewere bled 7 days after intramuscular injection, and protein expressionwas determined by assaying mouse sera. The results of SEAP expression inmouse as a function of N:P ratio are shown in FIG. 3E.

FIG. 3E shows the impact of N:P ratio and RNA dose on the bioactivity ofLION formulated repRNA.

Example 4. Using LION Formulations for Vaccine Delivery

This example shows that antigens expressed off of LION-complexed RNA arehighly immunogenic and induce antibodies. RNA molecules encoding avaccine antigen were complexed with a LION formulation, which weredelivered in the cytoplasm of cells of an organism. The RNA underwentintracellular translation and produced the vaccine antigen. The organismmounted an immune response by producing antibodies against the antigen.

A self-replicating “sr” RNA preparation, encoding a form of the spike“S” protein full-length, was mixed and formulated with LIONs. Mice wereimmunized once intramuscularly, with the formulated test articles at thedosage levels of 10, 1, and 0.1 μg of srRNA. At 14 dayspost-immunization, animals were bled, sera prepared and stored inaliquots at −20° C. until use. Antigen-specific IgG concentration usinga polyclonal IgG standard were determined against a truncatedreceptor-binding domain (RBD) protein fragment. The results are shown inFIG. 4A. As seen in FIG. 4A, the antigens expressed off of theLION/repRNA formulations at various doses of RNA (10, 1, and 0.1 μg,respectively) all induced strong and robust immune responses to thereceptor binding domain of SARS-CoV-2. Robust titers were seen even atvery low concentrations of RNA (0.1 μg).

Impact of the Size of the Core Inorganic Particles; LION Delivery Vs.NLC Delivery

RepSARS-CoV2S RNAs complexed with various LION formulations varying SPIOsize (LION-10, LION-15, LION-25, LION-5 respectively), or with ananostructured lipid carrier (NLC) as control, were administeredintramuscularly in C57BL/6 mice. The formulations labeled as LION-10,LION-15, LION-25, and LION-5 correspond to the LION compositions labeledas 79-004, 79-006-A, 79-006-B, 79-011, 79-014-A, respectively (see Table1 below; see also Example 1). Mice were bled at regular intervals afterintramuscular injection, and protein expression was determined byassaying IgG concentrations by anti-Spike (anti-S) enzyme linkedimmunosorbent assay (ELISA). The results of anti-S IgG concentrations inthe serum of the C57BL/6 mice over weeks post injection are shown inFIG. 4B.

FIG. 4B shows that LION formulations with the core inorganic SPIOnanoparticles having varying average diameters all worked to retain thebiological activity of antigens and induced robust levels of immuneresponse in vivo over a prolonged time period. Moreover, allRepSARS-CoV2S RNAs that were complexed with the LION formulationsgenerated responses greater than those complexed with the control NLC.

Impact of Mixing Direction and Dilution

RepSARS-CoV2S RNAs complexed with various LION formulations, prepared byvarying the mixing direction (mixing LION to RNA vs. mixing RNA to LION)and diluent (1:200 dilution using sucrose (Suc) vs. using dextrose(Dex)), were administered intramuscularly in C57BL/6 mice. Mice werebled at Day 14 (first bar for each group) or Day 21 (second bar for eachgroup) after intramuscular injection, and protein expression wasdetermined by assaying IgG concentrations by anti-Spike ELISA. Theresults are shown in FIG. 4C.

As shown in FIG. 4C, the direction of mixing (whether mixing LION to RNAor mixing RNA to LION) did not impact the biological activity ofantigens and the ability of the LION formulations to deliver the RNAs.Moreover, varying diluents (dextrose or sucrose) of the LION-RNAformulations did not impact the biological activity of antigens.

Example 5. Imaging LION Formulations

This example shows that LION formulations can emit signals for an MRIimaging. LION formulations complexed to RNA molecules or conjugated withmolecules to the nanoparticle surface or with molecules encapsulated inthe lipid core were administered to an organism. The organism wassubsequently placed in an imaging instrument and exposed toelectromagnetic waves, and the LION nanoparticles served to enhance thecontrast. The results are shown in FIGS. 5A-5B.

FIGS. 5A-5B show the enhancement in T1 (FIG. 5A) and T2 (FIG. 5B)relaxation times as a function of iron concentration in LIONformulations. The compositions of the LION formulations identified inFIGS. 5A-5B correspond to each of the LION compositions labeled as79-004, 79-006-A, 79-006-B, 79-011, 79-014-A, respectively (see Table 1below), according to Example 1, except that the concentrations of ironoxides in the LION composition were varies based on the X axis in thefigures.

FIG. 5 summarizes the ability to enhance both T1 and T2 contrast inmagnetic resonance imaging (MRI) using LION particles 79-004, 79-006-A,79-006-B, 79-011 and 79-014-A. The r1, r2 relaxivities and r2/r1 ratiosare summarized in Table 1.

TABLE 1 MR relaxivity (r1 and r2) and r2/r1 ratios of LION formulationscontaining iron oxides core with various core diameters. Formulation79-014- 79-004 79-006-A 79-006-B 79-011 A Alt. name LION/ LION-10LION-15 LION-5 LION-25 NLC-15 r1 [mM−1 s−1] 0.66 0.91 0.74 0.63 0.76 r2[mM−1 s−1] 1.12 1.59 0.46 2.47 1.60 r2/r1 1.70 1.75 0.63 3.94 2.10

Example 6. Using LION Formulations for Antibody Expression

This example shows that antibodies can be launched off of LION-complexRNA. RNA molecules encoding an antibody was complexed with a LIONformulation and delivered in the cytoplasm of cells of an organism. Themessenger RNA underwent intracellular translation and produced theantibody.

LION Formulation with a Replicon RNA Encoding a Monoclonal AntibodyTargeting Zika Virus

FIG. 6A shows robust levels of a human monoclonal antibody (ZIKV-117)that recognizes the Zika virus being produced after immunization ofanimals with the LION/antibody sequence RNA (at 40 μg RNA) formulation,varying SPIO size of the LION formulation (LION-10, LION-15, LION-25,LION-5 respectively). Animals were bled 7 days after immunization. Theresults indicate that LION formulations can be used to produceantibodies in a living organism. A nanostructured lipid carrier (NLC)formulated with the antibody sequence RNA was used as control. As shownin FIG. 6A, the antibody sequence RNA complexed with LION formulationsresulted in higher overall levels of ZIKV-117 expression with each SIPOsize, as compared to the antibody sequence RNA complexed with NLC. Thisindicates that compared to NLC, LION formulation served as a betterdelivery vehicle for antibody sequence RNAs.

LION Formulation with HIV and ZIKV Vaccine Candidates for MaternalImmunization in a Rabbit Model

FIGS. 6B and 6C show the magnitude and kinetics of anti-BG505 SOSIP.664IgG antibodies in adult female pregnant rabbits immunized byintramuscular route with saline (FIG. 6B) or repRNA encoding BG505SOSIP.664 trimer formulated with LION (FIG. 6C). FIGS. 6D and 6E showthe magnitude and kinetics of anti-ZIKV E IgG antibodies in adult femalepregnant rabbits immunized by intramuscular route with saline (FIG. 6D)or repRNA encoding ZIKV prM-E antigens formulated with LION (FIG. 6E).The shaded region around week 1 marks the period when rabbits were bred.The shaded region between weeks 6 and 7 marks the period when kits weredelivered. Arrows mark immunization time points (weeks 0, 4 and 11). Theresults show that the antigens expressed off of the LION/repRNAformulations induced strong and robust immune responses and producedstrong levels of antibodies for both LION/repRNA formulations.

FIGS. 6F and 6G show the results for the evaluation of in utero transferof anti-SOSIP IgG from rabbit does (female rabbits) to rabbit kits(rabbit babies). FIG. 6F shows anti-SOSIP IgG responses in rabbit kitsat time of delivery. A minimum of two rabbit kits from each litter pertreatment group were euthanized to evaluate in utero antibody transfer.FIG. 6G shows the XY plot demonstrating a positive correlation (Pearsonr=0.94) between antibody levels in rabbit does and corresponding rabbitkits. These figures demonstrated the material transfer of antibody fromthe breeding rabbit does to rabbit kits.

FIGS. 6H and 6I show the vaccine-induced responses in the context ofpre-existing maternal antibodies. Serum anti-SOSIP IgG levels werecollected in rabbit kits 4 weeks post-boost (3 weeks after kits wereweaned). The rabbit kits from rabbit does receiving saline or fromrabbit does receiving LION+RNA-prM/E are grouped as negative (−) forpre-existing maternal antibodies against BG505 SOSIP.664. The rabbitkits from rabbit does receiving LION+RNA-SOSIP or AddaVax adjuvantedrecombinant BG505 SOSIP.664 are grouped as positive (+) for pre-existingmaternal antibodies against BG505 SOSIP.664. Data show that pre-existingantibodies did not have a significant impact on vaccine-mediatedinduction of antibodies, and that passively transferred antibodies inrabbit kits did not negatively impact on vaccine-mediated induction ofantibodies in rabbit kits.

Example 7. Using LION Formulations for Vaccine Delivery in NonhumanPrimates—Single-Dose Replicating RNA Vaccine Induces NeutralizingAntibodies Against SARS-CoV-2 in Nonhuman Primates

This example discusses the development of repRNA-CoV2S, a stable andhighly immunogenic vaccine candidate comprising an RNA repliconformulated with a novel Lipid InOrganic Nanoparticle (LION) designed toenhance vaccine stability, delivery and immunogenicity.

Vaccine Design, Preparation, and Characterization

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratorysyndrome coronavirus-2 (SARS-CoV-2) infection, has been declared aworldwide pandemic. Coronaviruses are enveloped, single-strandpositive-sense RNA viruses with a large genome and open reading framesfor four major structural proteins: Spike (S), envelope, membrane, andnucleocapsid. The S protein mediates binding of coronaviruses toangiotensin converting enzyme 2 (ACE2) on the surface of various celltypes including epithelial cells of the pulmonary alveolus. Protectionmay be mediated by neutralizing antibodies against the S protein, asmost of the experimental vaccines developed against the related SARS-CoVincorporated the S protein, or its receptor binding domain (RBD), withthe goal of inducing robust, neutralizing responses. Previous reportshave shown that human-neutralizing antibodies protected mice, challengedwith SARS-CoV and Middle East respiratory syndrome (MERS)-CoV,suggesting that protection against SARS-CoV-2 may be mediated throughanti-S antibodies. Additionally, SARS vaccines that drive Type 2 Thelper (Th2) responses have been associated with enhanced lungimmunopathology following challenge with SARS-CoV, while those with aType 1 T helper (Th1)-biased immune response have been associated withenhanced protection in the absence of immunopathology. An effectiveCOVID-19 vaccine, therefore, may need to induce Th1-biased immuneresponses comprising SARS-CoV-2-specific neutralizing antibodies.

Nucleic acid vaccines have emerged as ideal modalities for rapid vaccinedesign, requiring only the target antigen's gene sequence and removingdependence on pathogen culture (inactivated or live attenuated vaccines)or scaled recombinant protein production. In addition, nucleic acidvaccines can avoid pre-existing immunity that can dampen immunogenicityof viral vectored vaccines. Clinical trials have been initiated withmessenger RNA (mRNA) vaccines formulated with lipid nanoparticles (LNPs)and a DNA vaccine delivered by electroporation. However, mRNA and DNAvaccines may not be able to induce protective efficacy in humans after asingle immunization, because, similar to inactivated and recombinantsubunit protein vaccines, they typically require multipleadministrations over an extended period of time to become effective.

Virus-derived replicon RNA (repRNA) vaccines were first described in1989 and have been delivered in the forms of virus-like RNA particles(VRP), in-vitro transcribed (IVT) RNA, and plasmid DNA. In repRNA, theopen reading frame encoding the viral RNA polymerase complex (mostcommonly from the Alphavirus genus) is intact but the structural proteingenes are replaced with an antigen-encoding gene. While conventionalmRNA vaccines are translated directly from the incoming RNA molecules,introduction of repRNA into cells initiates ongoing biosynthesis ofantigen-encoding RNA that results in dramatically increased expressionand duration that significantly enhances humoral and cellular immuneresponses. In addition, repRNA vaccines mimic an alphavirus infection inthat viral-sensing stress factors are triggered and innate pathways areactivated through Toll-like receptors and retinoic acid inducible gene(RIG)-I to produce interferons, pro-inflammatory factors and chemotaxisof antigen-presenting cells, as well as promoting antigen cross-priming.As a result, repRNA acts as its own adjuvant, eliciting more robustimmune responses after a single dose, relative to conventional mRNAwhich typically requires multiple and 1,000-fold higher doses.

Accordingly, repRNA vaccines were chosen as the vaccine candidates tostop a pandemic outbreak like COVID-19, as they have been studied withsome experiences, often require only a single administration to beeffective, and may have the potential of inducing protective levels ofimmunity rapidly with fewer and lower doses, while simultaneouslyreducing the load on manufacturing at scale.

As shown in FIG. 7A, repRNAs incorporating sequences from the SARS-CoV-2Spike (S) protein, including full length S (repRNA-CoV2S), weregenerated. Codon-optimized full length spike (S) open reading frame,including the S1-, S2-, transmembrane- (TM), and cytoplasmic- (CD)domains, corresponding to positions 21,536 to 25,384 in SARS-CoV-2isolate Wuhan-Hu-1 (GenBank: MN908947.3), fused to a c-terminal v5epitope tag, was cloned into an alphavirus replicon encoding the 4nonstructural protein (nsP1-4) genes of Venezuelan equine encephalitisvirus, strain TC-83. Following RNA transcription and capping,repRNA-COV2S, was transfected into BHK cells. Twenty four hours later,cells were analyzed by anti-v5 immunofluorescence and western blot usingeither convalescent human serum or anti-v5 for immunodetection, usingrecombinant SARS-CoV2 spike protein (rCoV2-Spike) and repRNA-GFP aspositive and negative controls, respectively. The results in FIGS. 7Band 7C show the efficient expression of the v5-tagged S protein in BHKcells. FIG. 7C also demonstrates the endogenous expression of an Sprotein in BHK cells, reactive with natural SARS-CoV-2 immune sera,utilizing convalescent serum collected 29 days after onset of COVID-19as an immunodetection reagent.

Formulation of repRNA-CoV2S with LION

Next, repRNA-CoV2S was formulated with an exemplary Lipid InOrganicNanoparticle (LION), designed to enhance vaccine stability andintracellular delivery of the vaccine. The ability of LION/repRNA-CoV2Sformulation to rapidly generate antibody and T cell responses wasevaluated in mice.

The general production techniques and materials for preparation of aLION composition followed those disclosed in Example 1. The exemplaryLION is a highly stable cationic squalene emulsion with 15 nmsuperparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIO), embedded inthe hydrophobic oil phase. FIG. 8A is a brief graphical representationof an exemplary LION and the formation of a vaccine complex after mixingLION with repRNA. Squalene is a vaccine adjuvant. SPIO nanoparticleshave clinical usage in MRI contrast and intravenous iron replacementtherapy; the unique nonlinear magnetic properties of SPIOs have alsoenabled their novel usages in a range of imaging, targeting and therapyapplications. The LION also contained the cationic lipid1,2-dioleoyl-3-trimethylammonium propane (DOTAP), which enabledelectrostatic association with RNA molecules when combined by a 1:1(v/v) mixing step. As disclosed in Example 1, this exemplary LION had anintensity-weighted average diameter of 52 nm (PDI=0.2), measured bydynamic light scattering (DLS). As shown in FIG. 8B, the LIONformulation was colloidally stable for at least 3 months when stored at4 and 25° C.

When mixing LION with repRNA, electrostatic association between anionicrepRNA and cationic DOTAP molecules on the surface of LION promotesimmediate complex formation. The formation of LION-repRNA complex wasconfirmed by the increase in particle size to an intensity-weightedaverage diameter of 90 nm, detected by DLS (see FIG. 8C). As shown inFIG. 8D, the gel electrophoresis analysis of LION-formulated repRNAmolecules extracted by phenol-chloroform treatment after a concentratedRNase challenge showed substantial protection from RNase-catalyzeddegradation, as compared to the unformulated repRNA (Naked). To evaluatethe short-term stability of the formulated vaccine, the repRNA integrityand complex stability were evaluated on 1, 4 and 7 days after mixing andstorage at 4° C. and 25° C., as determined by gel electrophoresis ofrepRNA extracted by phenol-chloroform treatment and particle size of thecomplex. As shown in FIGS. 8E and 8F, LION maintained full integrity ofthe repRNA molecules (FIG. 8E) and the complex maintained its size (FIG.8F) at all time points, indicating that the formulated vaccine complexwas stable for at least a week after mixing.

LION/repRNA-CoV2S Delivery in Mice

The LION/repRNA-CoV2S complex was administered to mice. Six toeight-week old C57BL/6 mice (n=5/group) received 10, 1, or 0.1 μgLION/repRNA-CoV2S via the intramuscular route. Fourteen days after primeimmunization, serum was harvested. As shown in FIG. 9A, a singleintramuscular immunization of C57BL/6 mice with 10 or 1 μg ofLION/repRNA-CoV2S induced 100% seroconversion by 14 dayspost-immunization and robust anti-S IgG levels with mean binding titersof 200 and 109 μg/ml, respectively, and partial seroconversion (2 out of5) at a 0.1 μg dose. As shown in FIG. 9B, both the 10 and 1 μgprime-only doses induced neutralizing antibodies with mean 50%inhibitory concentrations (IC50) of 1:643 and 1:226, respectively, asmeasured by pseudovirus neutralization assay (SARS-CoV-2 Wuhan-Hu-1pseudotype). While all doses induced Th1-biased immune responses, asindicated by significantly higher IgG2c responses when compared to IgG1(see FIG. 9C), there was a trend toward higher doses inducing even moreTh1-biased responses, as indicated by higher IgG2c:IgG1 ratios (see FIG.9D).

Given the potential role for T cells to contribute to protection, asseen with SARS and MERS, especially in the presence of waning antibodyand memory B cell responses, T cell responses to LION/repRNA-CoV2S werealso evaluated in mice. On day 28, this same cohort of mice received asecond immunization. Twelve days later, spleens and lungs were harvestedand stimulated with an overlapping 15-mer peptide library of the Sprotein, and the IFN-γ responses were measured by enzyme-linked immuneabsorbent spot (ELISpot) assay. As shown in FIG. 9E, the mice receivinga 10, 1, and 0.1 μg prime/boost exhibited robust splenic T cellresponses with mean IFN-γ spots/106 cells of 1698, 650, and 801,respectively. Robust T cell responses were also detected in the lung andwere similar between groups with mean IFN-γ spots/106 cells of 756, 784,and 777, respectively (see FIG. 9F).

The elderly are among the most vulnerable to COVID-19, but the immunesenescence in this population poses a barrier to an effectivevaccination. To evaluate the effect of immune senescence onimmunogenicity, 2-, 8-, or 17-month old BALB/C mice (n-5/group) received10 or 1 μg LION/repRNA-CoV2S via the intramuscular route. Fourteen daysafter the prime immunization, serum was harvested, and the anti-S IgGconcentrations were measured. As shown in FIG. 10A, significantly lowerantibody titers were observed in the 17-month old mice at both doses,when compared to the 2- and 8-month old mice, suggesting that higherdoses and/or additional booster doses may be needed in the mostimmune-senescent populations to induce sufficient immunity. Nodifferences were observed between the 2- and 8-month old mice. AlthoughBALB/C mice tend to develop a more Th2 immune-biased response followingvaccination, LION/repRNA-CoV2S induced the ratios of IgG2a:IgG1 ofgreater than 1 (see FIGS. 10B and 10C) in all age groups of the BALB/Cmice, indicating a Th1-biased immune response. Given that severe,life-threatening COVID-19 appears to be more common among the elderlyindividuals, irrespective of type of T helper response, and that severeSARS is associated with skewing toward Th2 antibody profiles with aninadequate Th1 response, the ability of LION/repRNA-CoV2S to inducestrong and Th1-biased responses in 8- and 2-month old mice, even in theTh2-biased BALB/c strain, provided positive signs regarding the safetyand immunogenicity of this vaccine complex.

LION/repRNA-CoV2S Delivery in Nonhuman Primates

Having achieved a robust immunogenicity with the LION/repRNA-CoV2Scomplex in mice, immunization of pigtail macaques (Macaca nemestrina)was then carried out to determine if the vaccine complex was capable ofinducing strong immune responses in a nonhuman primate model that moreclosely resembles humans in the immune response to vaccination.

In the dosage regime shown in FIG. 11A, three macaques received theLION/repRNA-CoV2S complex at a single 250 μg dose at week 0 via theintramuscular route, and two macaques received a 50 μg prime dose atweek 0 and a boost dose at week 4 via the intramuscular route. Blood wascollected 10, 14, 28, and 42 days post vaccination to monitor vaccinesafety and immunogenicity. The 50 μg group received a boost vaccinationon day 28, with the blood being collected 14 days later. There were noobserved reactions at the vaccine injection site nor adverse reactionsin the animals up to 42 days post-prime vaccination.

As shown in FIG. 11B, the ELISA analyses of sera collected 10, 14, 28,and 42 days after prime immunization, against the baseline establishedby the pre-immunization blood draws, showed that all three macaquesimmunized with the single 250 μg dose seroconverted as early as day 10,with anti-S IgG concentrations continuing to increase in these threeanimals to 48, 51, and 61 μg/ml by day 42. Both macaques receiving 50 μgrepRNA-CoV2S seroconverted after a single dose, but developedsignificantly lower antibody responses with anti-S IgG concentrations of1 and 0.5 μg/ml by day 28, as compared to 7, 20, and 45 μg/ml in the 250μg group at this same time point (see FIG. 11B). However, 14 days aftera booster immunization, the 50 μg group developed similar levels ofanti-S IgG concentrations (18 and 37 μg/ml) as the 250 μg prime-onlygroup at this time point (48, 51, and 61 μg/ml) (see FIG. 11B).Additionally, as shown in FIG. 11C, sera from the three macaquesimmunized with just the single 250 μg dose neutralized pseudovirus(SARS-CoV-2 Wuhan-Hu-1 pseudotype) transduction of cells in vitro withreciprocal IC50 titers of 1:38, 1:20 and 1:47 by day 28 with levelsincreasing to 1:472, 1:108, and 1:149 by day 42, whereas the 50 μg groupachieved similar robust IC50 titers only after the booster immunizationreaching pseudovirus IC50 titers of 1:218 and 1:358 by day 42.

Sera collected 28- and 42-days post vaccination were further analyzedfor neutralization of wild type SARS-CoV-2/WA/2020 by 80% plaquereduction neutralization test (PRNT80) and compared to neutralizingtiters in sera from convalescent humans collected 15-64 days followingnatural infection. As shown in FIG. 11D, a single immunization with 50and 250 μg of LION/repRNA-CoV2S induced mean PRNT80 titers of 1:32 and1:66 by day 28, respectively. By Day 42, mean PRNT80 titerssignificantly increased to 1:176 after a booster immunization in the 50μg group and to 1:211 in the prime-only 250 μg group. All 5 macaquesdeveloped PRNT80 titers within the same range as titers measured in theseven convalescent humans (<1:20 to 1:1280, collected 15 to 64 days postonset) and there was no significant difference in mean neutralizingtiters between all 5 vaccinated macaques (1:197) and convalescent humans(1:518) (P=0.27, FIG. 11D). Recently, serum-neutralizing titers,measured as the IC50 titer that neutralized SARS-CoV-2 by 50% tissueculture infectious dose (TCID50), were reported in rhesus macaques thatwere either re-infected or challenged after vaccination with aninactivated SARS-CoV-2 vaccine. In the former report, IC50 titers as lowas 1:8 were associated with protection from re-infection, while in thelatter, IC50 titers as low as 1:50 were associated with reduced viralload and protection from lung pathology. These data suggest that a 250μg prime-only or a 50 μg prime/boost immunization with theLION/repRNA-CoV2 vaccine would be able to induce levels of neutralizingantibodies sufficient to protect nonhuman primates from infection anddisease.

RepRNA vaccines against a variety of infectious diseases and cancershave been shown to be safe and potent in clinical trials, and thecell-free and potentially highly scalable manufacturing process ofrepRNA, when used with effective synthetic formulations, such as LION,presented further benefits over mRNA. The two-vial approach wouldprovide a significant manufacturing and distribution advantage over LNPformulations that encapsulate RNA, as the vaccine can be stockpiled andcombined onsite as needed. Additionally, the LION/repRNA-CoV-2 complexinduced robust S-specific T cell responses in mice. Following naturalinfection of humans with the related SARS-CoV, neutralizing antibody andmemory B cell responses in some individuals are reported to be shortlived (— 3 years) while memory T cells persist at least 6 years (53),suggesting a potential role for T cells in long term responsesespecially in those who lack robust memory B cell responses.Additionally, anti-S T-cell responses to the related SARS- and MERS-CoVscontribute towards viral clearance in normal as well as aged miceinfected with SARS- or MERS-CoV, respectively.

In sum, these results demonstrate a great potential for theLION/repRNA-CoV2S, complex to induce a rapid immune protection fromSARS-CoV-2 infection. A scalable and widely distributed vaccine capableof inducing robust immunity in both young and aged populations againstSARS-CoV-2 infection in a single shot would provide immediate andeffective containment of the pandemic. Critically, the vaccine inducedTh1-biased antibody and T cell responses in both young and aged mice, anattribute that has been associated with improved recovery and milderdisease outcomes in SARS-CoV-infected patients. A single-doseadministration in nonhuman primates elicited antibody responses thatpotently neutralized SARS-CoV-2. These data support the potential of theLION/repRNA-CoV2S complex as a vaccine for protection from SARS-CoV-2infection.

Example 8. Using LION Formulations for Vaccine Delivery in Rabbits

Animals: 8 New Zealand White rabbits (Oryctolagus cuniculus), 4 malesand 4 females, were separated into two groups, of each 2 males andfemales. Group 1 was injected with high dose LION-RNA formulation (250μg repRNA with LION formulation) and Group 2 was injected low doseLION-RNA formulation (10 μg repRNA with LION formulation).

Vaccine Preparation: LION carrier and repRNA-CoV2S were complexed at anitrogen-to-phosphate molar ratio of 15 in 10 mM sodium citrate and 20%sucrose buffer and were delivered to the study animals in three 0.5 mLintramuscular dosages, two weeks apart.

ELISA: Antigen-specific IgG responses were detected by ELISA usingrecombinant SARS-CoV-2S as the capture antigen. ELISA plates (Nunc,Rochester, NY) were coated with 1 μg/mL antigen or with serial dilutionsof purified polyclonal IgG to generate a standard curve in 0.1 M PBSbuffer and blocked with 0.2% BSA-PBS. Then, in consecutive order,following washes in PBS/Tween, serially diluted serum samples,anti-rabbit IgG-HRP (Southern Biotech, Birmingham, AL), and TMBperoxidase substrate were added to the plates, followed by quenchingwith HCl. Plates were analyzed at 405 nm (ELX808, Bio-Tek InstrumentsInc, Winooski, VT). Absorbance values from each serum dilution pointwere used to calculate titers.

FIG. 12 shows the anti-spike IgG levels in the rabbits injectedintramuscularly with repRNA-SARS-CoV2S (at 250 μg and 10 μg dose level,respectively) formulated with LION formulation. As shown in the example,animals rapidly mounted an immune response to the injected vaccine. Atweek 4 in both dose levels, antibody titers nearly plateaued and did notincrease significantly in weeks 6 or 8.

Example 9. Using LION Formulations for Vaccine Delivery— RSV Vaccine

RSV repRNA (2.5 μg) complexed with a LION formulation was administeredintramuscularly in C57Bl/6 and BALB/c mice. Mice blood was collected 28days after intramuscular injection, and protein expression wasdetermined by assaying Anti-F IgG concentrations by ELISA. The resultsof anti-F IgG levels in the serum of the C57Bl/6 and BALB/c mice areshown in FIG. 13A. As shown in FIG. 13A, RVS G protein-specificresponses were induced by replicon 646 in both C57BL/6 and BALB/c mice.

RSV repRNA (2.5 μg) complexed with a LION formulation was administeredintramuscularly in C57Bl/6 and BALB/c mice. Mice blood was collected 28days after intramuscular injection, and protein expression wasdetermined by assaying Anti-G (A2) IgG concentrations by ELISA. Theresults of anti-G (A2) IgG levels in the serum of the C57Bl/6 and BALB/cmice are shown in FIG. 13B. As shown in FIG. 13B, RVS G A2 strainprotein-specific responses were induced by replicon 645 in only BALB/cmice.

Example 10. Using LION Formulations for Delivery of Immunomodulating RNALION Formulations Protect Immunomodulating RNA PAMP from RNases

An RIG-I agonist, PAMP, was formulated with an exemplary LIONformulation (15 nm, similar to 79-006A in Example 1). The generalproduction techniques and materials for preparation of a LIONcomposition followed those disclosed in Example 1.

FIG. 14A-14B show the binding of PAMP to the LION formulation thatprovided protection from RNase challenge. FIG. 14A shows the gelelectrophoresis analysis of PAMP-LION complexes at various N:Pcomplexing ratios (0.04, 0.2, 1, 5, and 25, respectively) run on an RNAgel and was assessed for free RNA. As shown in FIG. 14A, free RNA wasnot present for PAMP-LION formulations at N:P ratio of 1, 5 and 25. FIG.14B shows the gel electrophoresis analysis of PAMP-LION complexes,following a challenge with RNase A, as compared to naked PAMP(unformulated PAMP). RNA was extracted from LION and run on an agarosegel to assess RNA degradation. The results show complexing of PAMP toLION protected the RNA from RNase-catalyzed degradation, as compared tothe unformulated RNA (Naked).

Immune Stimulation of a RIG-I Agonist, PAMP, Delivered by LION

This example illustrates the immune stimulation of the RIG-I agonist,PAMP, delivered by the LION formulation, when the PAMP-LION complex wasadded to A549-Dual cells. A549-Dual cells contain two reporterconstructs: the IFN-β promoter that drives the expression of SEAP, andthe IFIT2 promoter that drives the expression of luciferase.

PAMP was formulated with a LION formulation at various N:P complexingratios (0.5, 1.5, 4.5, 13.5, 40.5, and 121.5), and 3.7 ng PAMP/LION wasadded to A549-Dual cells. FIG. 15 shows the activation of the IFN-βpromoter and IFIT2 measured by SEAP activity and luciferase activity inthe supernatant, respectively, by the PAMP-LION complex as a function ofN:P ratio. The results show the innate immune stimulation of the RIG-Iagonist, PAMP, delivered by the PAMP-LION formulations at all the N:Pratios, for both the IFN-β promoter and the IFIT2 promoter, although theN:P ratio at 4.5-40.5 appeared to provide better immune stimulations forthe IFN-β promoter.

Immune Stimulation of a RIG-I Agonist and TLR3 Agonist Delivered by LION

This example illustrates the immune stimulation of a RIG-I agonist,PAMP, and a TLR3 agonist, Riboxxim, delivered by the LION formulation,when the RNA-LION complex was added to A549-Dual cells.

PAMP (a RIG-I agonist) or Riboxxim (a TLR3 agonist), unformulated (nakedcontrol) or formulated with a LION formulation at a N:P ratio of 8, wasadded to A549-Dual cells. FIGS. 16A and 16B show the activation of theIFN-β promoter (FIG. 16A) and IFIT2 (FIG. 16B) measured by SEAP activityand luciferase activity in the supernatant, respectively, by thePAMP-LION formulation or Riboxxim-LION formulation, as compared tounformulated RNA. The results show the innate immune stimulation of boththe RIG-I agonist, PAMP, and the TLR3 agonist, Riboxxim, delivered bycomplexing with the LION formulations worked to induce innate immuneactivation, triggering robust levels of reporter protein expression ascompared to their unformulated naked control.

FIG. 16C shows the activation of the IFIT2 by the Riboxxim-LIONformulation, as compared to unformulated Riboxxim, as a function of theRiboxxim dose level. The results show that, at all tested dose levels,the LION formulations complexed with Riboxxim induced a higher level ofIFIT2 activation as compared to its unformulated naked control, althoughthe Riboxxim-LION formulation with a higher dose level induced astronger IFIT2 activation.

This example illustrates the immune stimulation of the RIG-I agonist,PAMP, delivered by the LION formulation, when the PAMP-LION complex wasdelivered intranasally to C57BL/6 mice. PAMP was formulated with a LIONformulation at an N:P ratio of 8, and 0.2, 1, or 5 μg PAMP/LION wasdelivered into the nares of C57BL/6 mice. Eight hours later, nasalcavities and lungs of the mice were removed and immediately frozen, thenthe RNA was extracted and subjected to PCR for various target genes.

FIG. 16D shows the dose-dependent induction of innate immune genes inthe nasal cavity of treated mice compared to naïve controls. FIG. 16Eshows the activation of innate immune genes in the lungs of treatedmice. FIG. 16F shows that the mice maintained body weight when beingadministered the PAMP:LION formulation intranasally for 3 consecutivedays.

These results demonstrate that LION supported the delivery of bioactivePAMP by intranasal inoculation; at all tested dose levels, the LIONformulations complexed with PAMP upregulated the protein expression inthe nasal cavity and the lung when the formulation was deliveredintranasally to mice.

Example 11. Production of Lipid Inorganic Nanoparticles (LIONs) withAluminum Hydroxide Core Labelled as 108-011

These LIONs comprise 37.5 mg/ml squalene, 37 mg/ml Span® 60, 37 mg/mlTween® 80, 30 mg/ml DOTAP chloride, TOPO-coated Al(OOH) (Alhydrogel® 2%)particles at a target concentration of 1 mg Al/ml and 10 mM sodiumcitrate dihydrate. The LION particles were manufactured using thefollowing procedures.

In a 50 ml centrifuge tube, 10 ml of Alhydrogel® was added andcentrifuged at 300 rpm for 3 minutes. The supernatant water was removedand replaced with an equal amount of methanol. The particles werecentrifuged again at 300 rpm for 3 minutes and the methanol supernatantwas removed and replaced with an equal amount of methanol. Thisprocedure was repeated an additional two times to remove residual waterand to re-suspend the Alhydrogel® particles in 10 ml of methanol. Thezeta potential of Alhydrogel® dispersed in methanol was +11.5 mV. Tothis dispersion, 1 ml of 250 mg/ml trioctylphosphine oxide (TOPO) wasadded and the mixture was left overnight in an orbital shaker maintainedat 37° C. and 250 rotations per minute. This was done to coat a layer ofTOPO on the surface of Alhydrogel® by ligand exchange reaction. Theexcess TOPO in the dispersion was removed by washing with methanol. Thezeta potential of the TOPO-coated Al(OOH) particles was recorded to be+5 mV. The reduction in zeta potential indicates the surfacemodification of Alhydrogel® with TOPO was successful. This process wasdone to convert the hydrophilic surface of Alhydrogel® to hydrophobic,thus facilitating the miscibility of Alhydrogel® in the ‘oil’ phase ofLION. Methanol in the TOPO coated Al(OOH) dispersion was evaporated inthe fume hood for 45 minutes at 55 degree Celsius leaving a dry coat ofTOPO-Al(OOH) particles. To the dried TOPO-Al(OOH) particles, 3.7 gramsof Span® 60, 3.75 grams of squalene and 3.0 grams of DOTAP chloride wereadded to prepare the “oil” phase. The oil phase was sonicated 45 minutesin a water bath pre-heated to 65° C.

Separately, in a 1-liter glass bottle, the “aqueous” phase was preparedby adding 19.5 grams of Tween® 80 to 500 ml of 10 mM sodium citratedihydrate solution prepared with Milli-Q water. The aqueous phase wasstirred for 30 minutes to allow complete dissolution of Tween® 80. Aftercomplete dissolution of Tween® 80, 92 ml of the aqueous phase wastransferred to a 200 ml beaker and incubated in a water bath pre-heatedto 65° C.

To the heated oil phase, 92 ml of the pre-heated aqueous phase wasadded. The mixture was immediately emulsified using a VWR® 200homogenizer (VWR International) until a homogenous colloid with amilk-like appearance was produced. The colloid was subsequentlyprocessed by passaging the fluid through a Y-type interaction chamber ofa M110-P microfluidizer at 30,000 psi. The fluid was passaged 17 timesuntil the z-average hydrodynamic diameter, measured by dynamic lightscattering (Malvern Zetasizer Ultra), was 61.9 nm with a 0.24polydispersity index. The microfluidized LION sample was terminallyfiltered with a 200 nm pore-size polyethersulfone (PES) syringe filter.

Table 2 summarizes the size and PDI of the resulting Alum-LIONnanoparticles before and after complexing with alphavirus-derivedreplicon RNA molecules. Table 3 below summarizes the characteristics ofthe resulting Alum-LION nanoparticles.

TABLE 2 Size and PDI of Alum-LION before and after complexing withalphavirus-derived replicon RNA molecules. Values below are mean ofthree technical replicates. Size of Alum-LION Size of Alum-LION PDI ofAlum-LION PDI of Alum-LION before complexing after complexing beforecomplexing after complexing Size (nm) SD Size (nm) SD PDI SD PDI SD61.92 0.488 95.48 2.129 0.241 0.003 0.271 0.009

TABLE 3 Characterization of the Alum-LION formulation. Property MethodValue Particle size Dynamic Light Scattering; mean ± 61.92 ± 0.5(Z-average) SD of three technical replicates nm Size Dynamic LightScattering; mean ± 0.241 ± 0.003 distribution SD of three technicalreplicates (PDI) Zeta Dynamic Light Scattering; mean ± 34.98 ± 2.47potential SD of five technical replicates mV Aluminum Inductivelycoupled plasma- 598 ± 14 concentration optical emission spectroscopy;μg/ml mean ± SD of three sample replicates DOTAP Reversed phase-Highperformance 20.37 ± 0.57 concentration liquid chromatography; mean ± SDmg/ml of three sample replicates Squalene Reversed phase-Highperformance 26.38 ± 0.60 concentration liquid chromatography; mean ± SDmg/ml of three sample replicates

Example 12. RNA Delivery with Exemplary LION Formulations

A VEE replicon RNA containing the nLuc sequence in the subgenome wasdiluted to 6.4 ng/4 and complexed to LION at an N:P ratio of 15 for 30minutes on ice. Two types of LION formulations were used: one having the15-nm iron oxide (Fe₃O₄) nanoparticles (SPIO) as the core (similar to79-006A prepared according to Example 1), and the other having theTOPO-coated aluminum oxyhydroxide nanoparticles as the core, preparedaccording to Example 11.

The RNA:LION complex was diluted 1:10 in buffer (10% sucrose, 5 mMNaCitrate), and 50 μL (16 ng RNA) was added to wells of a 96-well platecontaining A549-Dual cells (Invivogen) in 150 μL Optimem. Cells weretransfected for 4 hours, the media replaced with complete Dulbecco'sModified Eagle Medium (DMEM) (containing 10% fetal bovine serum,L-glutamine, and Penicillin/Streptomycin), and incubated overnight at37° C. with 5% CO2. The following day, the media was removed and nLucexpression was assessed using the Nano-Glo Luciferase Assay System(Promega) according to the manufacturer's instructions. Plates were readusing a Spectramax i3 plate reader (Molecular Devices).

FIG. 17 shows the resulting in vitro protein expression from theRNA:LION complexes with replicon RNA encoding nLuc, using SPIO (Fe-LION)or TOPO-coated aluminum oxyhydroxide nanoparticles (Al-LION) as the coreof the LION formulation. The figure demonstrates that both LIONformulations containing either the iron oxide nanoparticles or aluminumoxyhydroxide nanoparticles as the cores provided successful in vitrodelivery of nLuc replicon, when the RNA was complexed with LIONformulation.

All references disclosed herein, including patent references andnon-patent references, are hereby incorporated by reference in theirentirety as if each was incorporated individually.

It is to be understood that the terminology used herein is for thepurpose of describing specific embodiments only and is not intended tobe limiting. It is further to be understood that unless specificallydefined herein, the terminology used herein is to be given itstraditional meaning as known in the relevant art.

References throughout this specification to “one embodiment” or “anembodiment” and variations thereof means that a particular feature,structure, or characteristic described in connection with the embodimentare included in at least one embodiment, and are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments. The abbreviation “e.g.” is used herein toindicate a non-limiting example, and is synonymous with the term “forexample.”

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, i.e., one or more,and the letter “s” following a noun designates both the plural andsingular forms of that noun, unless the content and context clearlydictates otherwise. It should also be noted that the conjunctive terms,“and” and “or” are generally employed in the broadest sense to include“and/or,” which is intended to encompass an embodiment that includes allof the associated items or ideas and one or more other alternativeembodiments that include fewer than all of the associated items orideas, unless the content and context clearly dictates inclusivity orexclusivity as the case may be.

In addition, where features or aspects of the invention are described interms of Markush groups, it is intended that the invention embraces andis also thereby described in terms of any individual member and anysubgroup of members of the Markush group, and Applicants reserve theright to revise the application or claims to refer specifically to anyindividual member or any subgroup of members of the Markush group.

Where a range of values is provided herein, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention. For example,any concentration range, percentage range, ratio range, or integer rangeprovided herein is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated. Also, any number range recited herein relating to anyphysical feature, such as polymer subunits, size or thickness, are to beunderstood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means ±20% of theindicated range, value, or structure, unless otherwise indicated.

What is claimed is:
 1. A composition, wherein the composition comprises:nucleic acids, wherein the nucleic acids comprise a sequence encodingfor a protein region; lipid nanoparticles, wherein the lipidnanoparticles are characterized as having a z-average diameter particlesize measurement of 20 nm to about 60 nm when measured using dynamiclight scattering, and wherein the lipid nanoparticles comprise: asurface comprising cationic lipids; and a hydrophobic core thatcomprises liquid oil, wherein lipids present in the hydrophobic core arein liquid phase at 25 degrees Celsius, wherein the nucleic acids arecomplexed to the cationic lipids to form nucleic acid-lipid nanoparticlecomplexes; and an adjuvant for innate immune stimulation.
 2. Thecomposition of claim 1, wherein the adjuvant for innate immunestimulation comprises squalene.
 3. The composition of claim 1, whereinthe adjuvant for innate immune stimulation is a double-stranded RNA, aprotein, a virus-like particle, a carbohydrate, a saponin, a viralfragment, or a cellular fragment.
 4. The composition of claim 1, whereinthe adjuvant for innate immune stimulation comprises a toll-likereceptor (TLR) agonist or a RIG-I agonist.
 5. The composition of claim4, wherein the TLR agonist comprises a TLR2 agonist, a TLR3 agonist, aTLR4 agonist, a TLR7 agonist, a TLR8 agonist, or a TLR9 agonist.
 6. Thecomposition of claim 1, wherein the sequence encoding for the proteinregion comprises a sequence encoding an antigen.
 7. The composition ofclaim 6, wherein the antigen is derived from a virus.
 8. The compositionof claim 6, wherein the antigen is derived from a cancer.
 9. Thecomposition of claim 1, wherein the nucleic acids further comprisesequence encoding an RNA polymerase complex region from an RNA virus.10. The composition of claim 9, wherein the RNA virus is a Venezuelanequine encephalitis virus.
 11. The composition of claim 1, wherein thecationic lipids comprise 1,2-dioleoyloxy-3-(trimethylammonium)propane(DOTAP); 3b-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DCCholesterol); dimethyldioctadecylammonium (DDA);1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP),dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP);distearoyltrimethylammonium propane (DSTAP);N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC);1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA); or 1,r-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C 12-200).
 12. The composition of claim 1, wherein the cationic lipidsare DOTAP.
 13. The composition of claim 1, wherein the hydrophobic corecomprises a solid inorganic nanoparticle.
 14. The composition of claim13, wherein the solid inorganic nanoparticle is selected from the groupconsisting of metal salts, metal oxides, metal hydroxides, and metalphosphates.
 15. The composition of claim 13, wherein the solid inorganicnanoparticle comprises iron oxide.
 16. The composition of claim 1,wherein the liquid oil is a vegetable oil, an animal oil, or asynthetically prepared oil.
 17. The composition of claim 1, wherein theliquid oil is capric/caprylic triglyceride, vitamin E, lauroylpolyoxylglyceride, monoacylglycerol, sunflower oil, soybean oil, oliveoil, grapeseed oil, or any combination thereof.
 18. The composition ofclaim 1, wherein the liquid oil is squalene.
 19. The composition ofclaim 1, wherein the lipid nanoparticles further comprise a hydrophobicsurfactant and a hydrophilic surfactant.
 20. The composition of claim18, wherein the hydrophobic surfactant is selected from the groupconsisting of sorbitan monostearate, sorbitan monooleate, and sorbitantrioleate.
 21. The composition of claim 18, wherein the hydrophilicsurfactant comprises polyethylene oxide.
 22. The composition of claim21, wherein the polyethylene oxide comprises polyoxyethylene sorbitanester.
 23. The composition of claim 19, wherein the hydrophilicsurfactant comprises polysorbate.
 24. The composition of claim 23,wherein the polysorbate comprises polysorbate
 80. 25. The composition ofclaim 1, wherein the cationic lipids comprise DOTAP, and wherein thelipid nanoparticles further comprise: squalene, sorbitan monostearate,and polysorbate
 80. 26. The composition of claim 25, wherein thecomposition further comprises sodium citrate.
 27. The composition ofclaim 1, wherein the nucleic acids comprise RNA.
 28. The composition ofclaim 27, wherein the RNA comprises mRNA.
 29. The composition of claim1, wherein the lipid nanoparticles are characterized as having az-average diameter particle size measurement of about 40 nm to 60 nmwhen measured using dynamic light scattering.
 30. The composition ofclaim 1, wherein the composition is in the form of a nanoemulsion.